6-acetylmorphine analogs, and methods for their synthesis and use

ABSTRACT

The present invention relates to novel 6-acetylmorphine analogs, and methods for their synthesis and use. Such analogs are designed to provide a convenient linkage chemistry for coupling under mild conditions to a suitable group on a target protein, polypeptide, solid phase or detectable label.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/105,299, filed Aug. 20, 2018, which is a divisional of U.S.patent application Ser. No. 15/640,781, filed Jul. 3, 2017, now U.S.Pat. No. 10,052,379, issued Aug. 21, 2018, which is a divisional of U.S.patent application Ser. No. 14/775,644, filed Sep. 11, 2015, now U.S.Pat. No. 9,694,069, issued Jul. 4, 2017, which is the U.S. nationalphase of International Application No. PCT/US2014/027585, filed Mar. 14,2014, which designated the United States and which claims priority toprovisional U.S. patent application No. 61/785,538, filed Mar. 14, 2013,and to provisional U.S. patent application No. 61/952,719, filed Mar.13, 2014, each of which are hereby incorporated in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel 6-acetylmorphine analogs usefulfor preparing conjugates comprising, inter alia, proteins, polypeptides,and labels; to conjugates comprising such 6-acetylmorphine analogs, andto methods for their synthesis and use.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

6-Monoacetylmorphine (6-MAM, also known as 6-acetylmorphine or 6-AM) isone of three active metabolites of heroin (diacetylmorphine), the othersbeing morphine and morphine-6-glucuronide. 6-AM is rapidly created fromheroin in the body, and then is either metabolized into morphine orexcreted in the urine. Since 6-AM is a unique metabolite to heroin,identification of 6-AM is considered to be definitive evidence of heroinuse. This is significant because on a urine immunoassay drug screen, thetest typically tests for morphine, which is a metabolite of a number oflegal and illegal opiates/opioids such as codeine, morphine sulfate, andheroin. 6-AM remains in the urine for no more than 24 hours so a urinespecimen must be collected soon after the last heroin use, but thepresence of 6-AM guarantees that heroin was in fact used as recently aswithin the last day.

In developing a binding assay for 6-AM, the artisan must consider thatsamples may contain these metabolites of opiates/opioids. Thus,immunogenic and label conjugates should be designed to present 6-AM soas to provide an assay with minimal cross-reactivity to morphine,morphine-3-glucuronide, morphine-6-glucuronide and other opioids.Analogs for use in preparing such conjugates should also be designed toprovide convenient attachment to various proteins, polypeptides, andlabels under mild conditions.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide novel 6-AM analogs, andmethods for their synthesis and use. Such analogs are preferablydesigned to provide a reactive thiol (—SH) group, providing a linkagechemistry for convenient coupling to a suitable group on a targetprotein, polypeptide, or label.

For purposes of the following discussion, the following depicts theposition numbering used in the art for morphine:

Thus, 6-AM has the following structure:

In a first aspect then, the invention relates to compounds (or saltsthereof) having a general formula selected from (I), (II), (III), or(IV):

whereR1, R3, R4, or R5 is a linkage chemistry which provides a terminalfunctional moiety selected from the group consisting of protected orunprotected sulfhydryl moieties, protected or unprotected aminemoieties, primary amine-reactive moieties, sulfhydryl-reactive moieties,photoreactive moieties, carboxyl-reactive moieties, arginine-reactivemoieties, and carbonyl-reactive moieties;

each Z is independently optionally substituted C₁₋₄ alkyl, C₁₋₄ alkoxy,N, O, S, and aryl, wherein substitution(s), when present, may beindependently selected from the group consisting of C₁₋₆ alkyl straightor branched chain, benzyl, halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂,—NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl, —CH₂OH, and —CONH₂,and where Z is preferably O, S, N, NH, CH₃, CH₂, CH, CHF or CF₂, andwhere Z is most preferably —C(O)—, —N(H)— or —O—;

each Y is independently selected from the group consisting of

wherein each R2 is independently optionally substituted C₁₋₄ alkyl, C₁₋₄alkoxy, OH, N, O, S, and aryl, wherein substitution(s), when present,may be independently selected from the group consisting of C₁₋₆ alkylstraight or branched chain, benzyl, halogen, trihalomethyl, C₁₋₆ alkoxy,—NO₂, —NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl, —CH₂OH, and—CONH₂, and where each R2 is preferably CH₃, CF₃, CHF₂, CH₂F or NH₂; andeach X is H or together form a covalent bond.Most preferably —Z—Y is

In certain embodiments, R1, R3, R4, or R5 is a linking group having thestructure Q-J, where Q is a linker that is saturated or unsaturated,substituted or unsubstituted, aromatic or aliphatic, straight orbranched chain of 0-10 carbon or heteroatoms (N, O, S), with an optionalC(O), S(O) or S(O₂); and J is a functional moiety selected from thegroup consisting of protected or unprotected sulfhydryl moieties,protected or unprotected amine moieties, primary amine-reactivemoieties, sulfhydryl-reactive moieties, photoreactive moieties,carboxyl-reactive moieties, arginine-reactive moieties, andcarbonyl-reactive moieties.

In certain preferred embodiments, R1, R3, R4, or R5 is a linking grouphaving the structure

whereW is C₀₋₄ unsubstituted alkyl;X is an optionally present C(O);Y is an optionally substituted C₀₋₄ alkyl or N(H)—C₀₋₆ alkyl, and isoptionally present; andZ is a functional moiety selected from the group consisting of protectedor unprotected sulfhydryl moieties, protected or unprotected aminemoieties, primary amine-reactive moieties, sulfhydryl-reactive moieties,photoreactive moieties, carboxyl-reactive moieties, arginine-reactivemoieties, and carbonyl-reactive moieties.

The choice of functional moiety may be varied by the artisan, dependingon the desired length and composition for a crossbridge to a protein,polypeptide or label, and whether the functional moiety is in free or inprotected form. In the latter case, a wide variety of protective groupsfor such functional moieties are known in the art. See, e.g., standardreference works such as Greene and Wuts, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, 3^(rd) edition, John Wiley & Sons Inc., 1999, which is herebyincorporated by reference in its entirety. By way of example only,suitable thiol protective groups include thioesters, thioethers,unsymmetrical disulfides, and sulfenyls.

In preferred embodiments, the functional moiety is a 5- or 6-membercyclic thiolactone, an optionally substituted C₁₋₄ alkyl thiol, or anoptionally substituted thioester having the structure

where R6 is selected from the group consisting of optionally substitutedC₁₋₄ alkyl, C₁₋₄ alkoxy, and aryl, wherein substitution(s), whenpresent, may be independently selected from the group consisting of C₁₋₆alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C₁₋₆alkoxy, —NO₂, —NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl,—CH₂OH, and —CONH₂.

In a related aspect, the invention relates to compositions comprisingone or more of the foregoing compounds (or their salts) covalentlylinked through the terminal functional moiety provided by R1 R3, or R4to a protein, polypeptide, label, or other molecule, referred to hereinas “6-AM analog conjugates.”

In this aspect, the invention relates to compounds (or salts thereof)having a general formula selected from (V), (VI), (VII) or (VIII):

whereR7, R8, R9, or R10 is a linkage chemistry and P is a protein,polypeptide, label, or other molecule, wherein R7, R8, R9, or R10 and Pare covalently linked;each Z is independently optionally substituted C₁₋₄ alkyl, C₁₋₄ alkoxy,N, O, S, and aryl, wherein substitution(s), when present, may beindependently selected from the group consisting of C₁₋₆ alkyl straightor branched chain, benzyl, halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂,—NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl, —CH₂OH, and —CONH₂,and where Z is preferably O, S, N, NH, CH₃, CH₂, CH, CHF or CF₂, andwhere Z is most preferably —C(O)—, —N(H)— or —O—.each Y is independently selected from the group consisting of

wherein each R2 is independently optionally substituted C₁₋₄ alkyl, C₁₋₄alkoxy, OH, N, O, S, and aryl, wherein substitution(s), when present,may be independently selected from the group consisting of C₁₋₆ alkylstraight or branched chain, benzyl, halogen, trihalomethyl, C₁₋₆ alkoxy,—NO₂, —NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl, —CH₂OH, and—CONH₂, and where each R2 is preferably CH₃, CF₃, CHF₂, CH₂F or NH₂; andeach X is H or together form a covalent bond.Most preferably each R2 is methyl and Z is N(H), such that —Z—Y is.

In certain embodiments, R7, R8, R9, or R10 is a linking group having thestructure Q-J, where Q is a linker that is saturated or unsaturated,substituted or unsubstituted, aromatic or aliphatic, straight orbranched chain of 0-10 carbon or heteroatoms (N, O, S), with an optionalC(O), S(O) or S(O₂); and J is a functional moiety conjugated to P via alinkage chemistry selected from the group consisting of sulfhydrylmoieties, amine moieties carboxyl moieties, arginine moieties, andcarbonyl moieties.

In certain preferred embodiments, R7, R8, R9, or R10 is a linking grouphaving the structure

whereW′ is C₀-4 unsubstituted alkyl;X′ is an optionally present C(O);Y′ is an optionally substituted C₀₋₄ alkyl or N(H)—C₀₋₆ alkyl, and isoptionally present; andZ′ is a functional moiety selected from the group consisting ofprotected or unprotected sulfhydryl moieties, protected or unprotectedamine moieties, primary amine-reactive moieties, sulfhydryl-reactivemoieties, photoreactive moieties, carboxyl-reactive moieties,arginine-reactive moieties, and carbonyl-reactive moieties.

The compounds of the present invention may be directly linked to anappropriate target protein, polypeptide, label, or other molecule toform a conjugate via a coupling group naturally occurring in the targetmolecule, or by adding a coupling group to the target molecule.Exemplary coupling groups are described hereinafter, and methods forincorporating such coupling groups into target molecules for conjugationto the compounds described above are well known in the art. In the caseof compounds of the invention comprising a protected functional moiety,removal of the protective group is performed by methods known in theart.

Preferred coupling groups on target molecules are maleimides, which arelinked according to the following reaction scheme:

where R—SH is a compound of the invention comprising a free thiol(either as a free thiol or following deprotection of a protected thiol),L is a linkage chemistry, and P is a target protein, polypeptide, label,or other molecule. L is preferably C₁₋₁₀ alkylene straight or branchedchain comprising from 0-4 backbone (i.e., non-substituent) heteroatoms,optionally substituted with from 1 to 4 substituents independentlyselected from the group consisting of C₁₋₆ alkyl straight or branchedchain, —NO₂, —NH₂, ═O, halogen, trihalomethyl, C₁₋₆ alkoxy, —OH, —CH₂OH,and —C(O)NH₂.

In certain embodiments, P is a protein, most preferably an antigenicprotein which can be used to raise an immune response to an epitope onthe compound of the invention using a so-called “hapten-carrier”immunogen. Common carrier proteins include bovine serum albumin, keyholelimpet hemocyanin, ovalbumin, etc. Protocols for conjugation of haptensto carrier proteins may be found in ANTIBODIES: A LABORATORY MANUAL, E.Harlow and D. Lane, eds., Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y., 1988) pp. 78-87, which is hereby incorporated byreference.

Alternatively, P may preferably be a detectable label. Preferreddetectable labels may include molecules or larger structures that arethemselves detectable (e.g., fluorescent moieties, electrochemicallabels, metal chelates, latex particles, etc.), as well as moleculesthat may be indirectly detected by production of a detectable reactionproduct (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, avidin, streptavidin, digoxigenin, maltose,oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).Exemplary conjugation to such detectable labels is describedhereinafter. Particularly preferred detectable labels are fluorescentlatex particles.

The foregoing lists of suitable target molecules are not meant to belimiting. Further exemplary embodiments are described hereinafter. Inaddition, numerous other classes of suitable targets, including peptidehormones, therapeutic proteins, antibodies, antibody fragments,single-chain variable region fragments, small molecules, nucleic acids,oligosaccharides, polysaccharides, cyclic polypeptides, peptidomimetics,aptamers and solid phases are known in the art.

While a conjugation target may be conjugated 1:1 with a 6-AM analog ofthe invention, an individual target may also comprise more than 1conjugation site, and hence more than 1 compound of the invention may beconjugated thereto. In preferred embodiments, a conjugation target(e.g., a protein, peptide, or label) comprises at least 10 6-AM analogmoieties covalently bound thereto, more preferably at least 30, stillmore preferably at least 50, and most preferably at least 100.

In still other related aspects, the present invention relates to methodsfor the production and use of the 6-AM analogs of the present inventionto form conjugates with a protein, polypeptide, label, or othermolecule.

Such methods can comprise contacting one or more compounds of theinvention comprising a reactive moiety (e.g., comprising a free thiol)with one or more target molecules comprising one or more correspondingcoupling sites, under conditions where the reactive moiety(s) react withthe coupling site(s) to form one or more conjugates. Conditions for suchreactions are dependent upon the reactive moiety(s) selected, and arewell known to the skilled artisan. Exemplary conditions are describedhereinafter.

Such methods may further comprise the step of deprotecting a protectedreactive moiety from one or more compounds of the invention prior tosaid contacting step, and/or attaching one or more coupling sites to aprotein, polypeptide, label, or other molecule to form an appropriateconjugation target. In the latter case, this may comprise the use ofbifunctional cross-linkers that provide an appropriate coupling sites atone site in the molecule, and a second coupling group for attachment tothe protein, polypeptide, label, or other molecule of interest. Numerousbifunctional cross-linkers are well known to those of skill in the art.

Regarding the use of such 6-AM analog conjugates, the present inventionrelates to methods for preparing an antibody. These methods compriseusing one or more conjugates as an immunogen to stimulate an immuneresponse.

In certain embodiments, methods may comprise administering one or moreconjugates of the invention in a suitable immunization protocol, andseparating an appropriate antibody from a body fluid of the animal.Exemplary protocols for preparing immunogens, immunization of animals,and collection of antiserum may be found in ANTIBODIES: A LABORATORYMANUAL, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory (ColdSpring Harbor, N.Y., 1988) pp. 55-120, which is hereby incorporated byreference. Alternatively, the 6-acetylmorphine analog conjugates of thepresent invention may be used in phage display methods to select phagedisplaying on their surface an appropriate antibody, followed byseparation of nucleic acid sequences encoding at least a variable domainregion of an appropriate antibody. Phage display methods are well knownto those of skill in the art. Such methods may use immunized orunimmunized animals as a source of nucleic acids to form the phagedisplay library. Antibodies prepared in this manner may preferably finduse as therapeutic molecules and/or as receptors in receptor bindingassays.

Preferably, such antibodies bind 6-AM with an affinity that is at leasta factor of 5, more preferably at least a factor of 10, still morepreferably at least a factor of 30, and most preferably at least afactor of 50 or more, than an affinity for morphine,morphine-3-glucuronide, and/or morphine-6-glucuronide.

Antibodies prepared in this manner may be used as specific bindingreagents in immuoassays for determining 6-AM concentrations in samples.By way of example, a method can comprise performing a competitiveimmunoassay in using a conjugate having a general formula selected from(IV), (V), or (VI) in which P is a detectable label, the methodcomprising determining the concentration of 6-AM in the sample from theassay signal. Preferably, immunoassays provide a signal that is at leasta factor of 5, more preferably at least a factor of 10, still morepreferably at least a factor of 30, and most preferably at least afactor of 50 or more for 10 μg/mL 6-AM, compared to the signal obtainedfrom 10 μg/mL, and more preferably 1000 μg/mL, morphine,morphine-3-glucuronide, and/or morphine-6-glucuronide.

Other embodiments of the invention will be apparent from the followingdetailed description, exemplary embodiments, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A through 1E depict exemplary 6-AM analogs of the presentinvention.

FIG. 2 depicts reaction schemes to prepare exemplary 6-AM analogs of thepresent invention.

FIG. 3 depicts depict reaction schemes to prepare exemplary 6-AM analogsof the present invention.

FIG. 4 depicts a reaction scheme to prepare exemplary 6-AM analogs ofthe present invention.

FIG. 5 depicts a reaction scheme to prepare exemplary 6-AM analogs ofthe present invention.

FIG. 6 depicts a reaction scheme to prepare exemplary 6-AM analogs ofthe present invention.

FIG. 7 depicts an assay performance curve generated using multiple lotsof 6-AM antigen immunoconjugate against varying concentrations of 6-AM

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to 6-AM analogs and methods fortheir production and use, particularly for preparing cross-linkablethiol-containing 6-AM analogs for conjugation to another molecule, andfor use of such conjugates for preparing reagents for immunoassays thatdetect 6-AM. The analogs of the present invention are particularly wellsuited for producing antibodies and labels for use in receptor bindingassays for 6AM that can distinguish 6-AM from morphine,morphine-3-glucuronide, morphine-6-glucuronide and other opioids.

For the sake of clarity, definitions for the following terms regardingthe compounds of the present invention are provided.

As used herein, the term “aryl” refers to an optionally substitutedaromatic group with at least one ring having a conjugated pi-electronsystem, containing up to two conjugated or fused ring systems. Arylincludes carbocyclic aryl, heterocyclic aryl and biaryl groups, all ofwhich may be optionally substituted. Preferably, the aryl is eitheroptionally substituted phenyl, optionally substituted pyridyl,optionally substituted benzothiopyranyl, optionally substitutedcarbazole, optionally substituted naphthyl, optionally substitutedtetrahydronaphthyl. While “aryl” is most preferably a monocycliccarbocyclic aromatic ring having 5 or 6 ring atoms (and is mostpreferably phenyl), the aryl or heteroaryl Ar group (formed into anarylene or heteroarylene in the crosslinkers described herein byelaboration from a ring atom) generally may contain up to ten ringatoms, although the skilled artisan will recognize that aryl groups withmore than ten ring atoms are within the scope of the invention. The ringsystems encompassed by Ar can contain up to four heteroatoms,independently selected from the group consisting of N, S, and O.

Monocyclic aryl groups include, but are not limited to: phenyl,thiazoyl, furyl, pyranyl, 2H-pyrrolyl, thienyl, pyrroyl, imidazoyl,pyrazoyl, pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl moieties.Fused bicyclic Ar groups include, but are not limited to: benzothiazole,benzimidazole, 3H-indolyl, indolyl, indazoyl, purinyl, quinolizinyl,isoquinolyl, quinolyl, phthalizinyl, naphthyridinyl, quinazolinyl,cinnolinyl, isothiazolyl, quinoxalinyl indolizinyl, isoindolyl,benzothienyl, benzofuranyl, isobenzofuranyl, and chromenyl moieties.

As used herein, the term “heteroatom” refers to non-carbon, non-hydrogenatoms such as N, O, and S.

The aryl group may also be optionally substituted by replacement of oneor more hydrogen atoms by another chemical moiety. Preferredsubstituents include C₁₋₆ alkyl straight or branched (e.g. isopropyl)chain, halogen, trihalomethyl, alkoxy, NO₂, NH₂, OH, —COOR′, where R′ isH or lower alkyl, CH₂OH, and CONH₂.

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms. More preferably,it is a medium alkyl (having 1 to 10 carbon atoms). Most preferably, itis a lower alkyl (having 1 to 4 carbon atoms). The alkyl group may besubstituted or unsubstituted.

As used herein, the term “alkoxy” group refers to both an —O-alkyl andan —O-cycloalkyl group; preferably an alkoxy group refers to a loweralkoxy, and most preferably methoxy or ethoxy.

As used herein, the term “thiolactone” refers to a cyclic hydrocarbonhaving 5 or 6 ring atoms, one of which is an S heteroatom, and where theheteroatom is adjacent to a carbon substituted with a ═O.

As used herein, the term “thioester” refers to an organic compoundhaving the structure R—S—C(O)—R′.

As used herein, the term “alkyl thiol” refers to an alkyl groupcontaining an —SH group. Thiols are also referred to as “thio alcohols”and “sulfhydryls.”

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3^(rd) Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH₁ domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH₁ domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

The term “polypeptide” as used herein refers to a molecule having asequence of amino acids linked by peptide bonds. This term includesproteins, fusion proteins, oligopeptides, cyclic peptides, andpolypeptide derivatives. Antibodies and antibody derivatives arediscussed above in a separate section, but antibodies and antibodyderivatives are, for purposes of the invention, treated as a subclass ofthe polypeptides and derivatives. The term protein refers to apolypeptide that is isolated from a natural source, or produced from anisolated cDNA using recombinant DNA technology, and that has a sequenceof amino acids having a length of at least about 200 amino acids.

The term “nucleic acids” as used herein shall be generic topolydeoxyribonucleotides (containing 2′-deoxy-D-ribose or modified formsthereof), to polyribonucleotides (containing D-ribose or modified formsthereof), and to any other type of polynucleotide which is anN-glycoside of purine or pyrimidine bases, or modified purine orpyrimidine bases.

The term “aptamer” as used herein is a single-stranded ordouble-stranded oligodeoxyribonucleotide, oligoribonucleotide ormodified derivatives that specifically bind and alter the biologicalfunction of a target molecule. The target molecule is defined as aprotein, peptide and derivatives thereof. The aptamer is capable ofbinding the target molecule under physiological conditions. An aptamereffect is distinguished from an antisense effect in that the aptamericeffects are induced by binding to the protein, peptide and derivativethereof and are not induced by interaction or binding underphysiological conditions with nucleic acid.

The term “polysaccharide” as used herein refers to a molecule comprisingmore than 10 glycosidically linked monosaccharide residues, while theterm “oligosaccharide” refers to a molecule comprising from 2-10glycosidically linked monosaccharide residues.

The term “small molecule” includes any molecule having a molecularweight less than about 5,000 daltons (Da), preferably less than about2,500 Da, more preferably less than 1,000 Da, most preferably less thanabout 500 Da.

Functional Moieties

Chemical cross-linkers are valuable tools for preparingantibody-detectable label conjugates, immunotoxins and other labeledprotein and nucleic acid reagents. These reagents may be classified onthe basis of the following:

1. Functional groups and chemical specificity;

2. length and composition of the cross-bridge;

3. whether the cross-linking groups are similar (homobifunctional) ordifferent (heterobifunctional);

4. whether the groups react chemically or photochemically;

5. whether the reagent is cleavable; and

6. whether the reagent can be radiolabeled or tagged with another label.

As the compounds of the present invention provide an available thiol toact as an attachment point, targets may be prepared to provide anappropriate thiol-reactive site. Cross-linking reagents that couplethrough sulfhydryls (thiols) are available from many commercial sources.Maleimides, alkyl and aryl halides, and alpha-haloacyls react withsulfhydryls to form thiol ether bonds, while pyridyl disulfides reactwith sulfhydryls to produce mixed disulfides. The pyridyl disulfideproduct is cleavable. Such reagents may be bifunctional, in that asecond site on the reagent is available for use in modifying aconjugation target to incorporate the thiol-reactive site. In additionto thiols, reactive groups that can be targeted using a cross-linkerinclude primary amines, carbonyls, carbohydrates and carboxylic acids.In addition, many reactive groups can be coupled nonselectively using across-linker such as photoreactive phenyl azides. Thus, a two-stepstrategy allows for the coupling of a protein that can tolerate themodification of its amines to a 6-acetylmorphine analog of theinvention. For suitable reagents, see Pierce 2003-2004 ApplicationsHandbook and Catalog #1600926, which is hereby incorporated byreference. Cross-linkers that are amine-reactive at one end andsulfhydryl-reactive at the other end are quite common. If usingheterobifunctional reagents, the most labile group is typically reactedfirst to ensure effective cross-linking and avoid unwantedpolymerization.

Many factors must be considered to determine optimumcross-linker-to-target molar ratios. Depending on the application, thedegree of conjugation is an important factor. For example, whenpreparing immunogen conjugates, a high degree of conjugation is normallydesired to increase the immunogenicity of the antigen. However, whenconjugating to an antibody or an enzyme, a low-to-moderate degree ofconjugation may be optimal to ensure that the biological activity of theprotein is retained. It is also important to consider the number ofreactive groups on the surface of the protein. If there are numeroustarget groups, a lower cross-linker-to-protein ratio can be used. For alimited number of potential targets, a higher cross-linker-to-proteinratio may be required. This translates into more cross-linker per gramfor a small molecular weight protein.

Conformational changes of proteins associated with a particularinteraction may also be analyzed by performing cross-linking studiesbefore and after the interaction. A comparison is made by usingdifferent arm-length cross-linkers and analyzing the success ofconjugation. The use of cross-linkers with different reactive groupsand/or spacer arms may be desirable when the conformation of the proteinchanges such that hindered amino acids become available forcross-linking.

Cross-linkers are available with varying lengths of spacer arms orbridges connecting the reactive ends. The most apparent attribute of thebridge is its ability to deal with steric considerations of the moietiesto be linked. Because steric effects dictate the distance betweenpotential reaction sites for cross-linking, different lengths of bridgesmay be considered for the interaction. Shorter spacer arms are oftenused in intramolecular cross-linking studies, while intermolecularcross-linking is favored with a cross-linker containing a longer spacerarm.

The inclusion of polymer portions (e.g., polyethylene glycol (“PEG”)homopolymers, polypropylene glycol homopolymers, otheralkyl-polyethylene oxides, bis-polyethylene oxides and co-polymers orblock co-polymers of poly(alkylene oxides)) in cross-linkers can, undercertain circumstances be advantageous. See, e.g., U.S. Pat. Nos.5,643,575, 5,672,662, 5,705,153, 5,730,990, 5,902,588, and 5,932,462;and Topchieva et al., Bioconjug. Chem. 6: 380-8, 1995). For example,U.S. Pat. No. 5,672,662 discloses bifunctional cross-linkers comprisinga PEG polymer portion and a single ester linkage. Such molecules aresaid to provide a half-life of about 10 to 25 minutes in water.

Designing a cross-linker involves selection of the functional moietiesto be employed. The choice of functional moieties is entirely dependentupon the target sites available on the species to be crosslinked. Somespecies (e.g., proteins) may present a number of available sites fortargeting (e.g., lysine ε-amino groups, cysteine sulfhydryl groups,glutamic acid carboxyl groups, etc.), and selection of a particularfunctional moiety may be made empirically in order to best preserve abiological property of interest (e.g., binding affinity of an antibody,catalytic activity of an enzyme, etc.)

1. Coupling Through Amine Groups

Imidoester and N-hydroxysuccinimidyl (“NHS”) esters are typicallyemployed as amine-specific functional moieties. NHS esters yield stableproducts upon reaction with primary or secondary amines. Coupling isefficient at physiological pH, and NHS-ester cross-linkers are morestable in solution than their imidate counterparts. HomobifunctionalNHS-ester conjugations are commonly used to cross-link amine-containingproteins in either one-step or two-step reactions. Primary amines arethe principle targets for NHS-esters. Accessible α-amine groups presenton the N-termini of proteins react with NHS-esters to form amides.However, because α-amines on a protein are not always available, thereaction with side chains of amino acids become important. While fiveamino acids have nitrogen in their side chains, only the ε-amino groupof lysine reacts significantly with NHS-esters. A covalent amide bond isformed when the NHS-ester cross-linking agent reacts with primaryamines, releasing N-hydroxysuccinimide.

2. Coupling Through Sulfhydryl Groups

Maleimides, alkyl and aryl halides, α-haloacyls, and pyridyl disulfidesare typically employed as sulfhydryl-specific functional moieties. Themaleimide group is specific for sulfhydryl groups when the pH of thereaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reactionof the maleimides with sulfhydryls is 1000-fold faster than with amines.Maleimides do not react with tyrosines, histidines or methionines. Whenfree sulfhydryls are not present in sufficient quantities, they canoften be generated by reduction of available disulfide bonds.

3. Coupling Through Carboxyl Groups

Carbodiimides couple carboxyls to primary amines or hydrazides,resulting in formation of amide or hydrazone bonds. Carbodiimides areunlike other conjugation reactions in that no cross-bridge is formedbetween the carbodiimide and the molecules being coupled; rather, apeptide bond is formed between an available carboxyl group and anavailable amine group. Carboxy termini of proteins can be targeted, aswell as glutamic and aspartic acid side chains. In the presence ofexcess cross-linker, polymerization may occur because proteins containboth carboxyls and amines. No cross-bridge is formed, and the amide bondis the same as a peptide bond, so reversal of the cross-linking isimpossible without destruction of the protein.

4. Nonselective Labeling

A photoaffinity reagent is a compound that is chemically inert butbecomes reactive when exposed to ultraviolet or visible light.Arylazides are photoaffinity reagents that are photolyzed at wavelengthsbetween 250-460 nm, forming a reactive aryl nitrene. The aryl nitrenereacts nonselectively to form a covalent bond. Reducing agents must beused with caution because they can reduce the azido group.

5. Carbonyl Specific Cross-Linkers

Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH5-7. The reaction with hydrazides is faster than with amines, makingthis useful for site-specific cross-linking. Carbonyls do not readilyexist in proteins; however, mild oxidation of sugar moieties usingsodium metaperiodate will convert vicinal hydroxyls to aldehydes orketones.

Exemplary Applications for Use of Cross-Linkable 6-AcetylmorphineAnalogs

1. Carrier Protein-Hapten/Peptide/Polypeptide Conjugates for Use asImmunogens

Numerous companies offer commercially available products in this area ofimmunological research. There are many cross-linkers used for theproduction of these conjugates, and the best choice is dependent on thereactive groups present on the hapten and the ability of thehapten-carrier conjugate to function successfully as an immunogen afterits injection. Carbodiimides are good choices for producing peptidecarrier conjugates because both proteins and peptides usually containseveral carboxyls and primary amines. Other cross-linkers can also beused to make immunogen conjugates.

Adjuvants are mixtures of natural or synthetic compounds that, whenadministered with antigens, enhance the immune response. Adjuvants areused to (1) stimulate an immune response to an antigen that is notinherently immunogenic, (2) increase the intensity of the immuneresponse, (3) preferentially stimulate either a cellular or a humoralresponse (i.e., protection from disease versus antibody production).Adjuvants have four main modes of action: enhanced antigen uptake andlocalization, extended antigen release, macrophage activation, and T andB cell stimulation. The most commonly used adjuvants fall into sixcategories: mineral salts, oil emulsions, microbacterial products,saponins, synthetic products and cytokines. A more extensive discussionof adjuvants and their use in immunization protocols is given inIMMUNOLOGY METHODS MANUAL, vol. 2, I. Lefkovits, ed., Academic Press,San Diego, Calif., 1997, ch. 13, which is hereby incorporated in itsentirety

Small molecules such as 6-acetylmorphine are not usually immunogenic,even when administered in the presence of adjuvant. In order to generatean immune response to these compounds, it is often necessary to attachthem to a protein or other compound, termed a carrier, that isimmunogenic. When attached to a carrier protein the small moleculeimmunogen is called a hapten. Haptens are also conjugated to carrierproteins for use in immunoassays. The carrier protein provides a meansof attaching the hapten to a solid support such as a microtiter plate ornitrocellulose membrane. When attached to agarose they may be used forpurification of the anti-hapten antibodies. They may also be used tocreate a multivalent antigen that will be able to form largeantigen-antibody complexes. When choosing carrier proteins, rememberthat the animal will form antibodies to the carrier protein as well asto the attached hapten. It is therefore important to select a carrierprotein for immunization that is unrelated to proteins that may be foundin the assay sample. If haptens are being conjugated for bothimmunization and assay, the two carrier proteins should be as differentas possible. This allows the antiserum to be used without having toisolate the anti-hapten antibodies from the anti-carrier antibodies.

Keyhole limpet hemocyanin (KLH) is a respiratory protein found inmollusks. Its large size makes it very immunogenic, and the large numberof lysine residues available for conjugation make it very useful as acarrier for haptens such as 6-acetylmorphine. The phylogenic separationbetween mammals and mollusks increases the immunogenicity and reducesthe risk of cross-reactivity between antibodies against the KLH carrierand naturally occurring proteins in mammalian samples.

2. Solid-Phase Immobilization

The analogs and/or conjugates of the present invention can beimmobilized on solid-phase matrices for use as affinity supports or forsample analysis. Similarly, antibodies or their binding fragments madeor selected using the 6-acetylmorphine analogs and/or conjugates of thepresent invention can also be immobilized on solid-phase matrices. Theterm “solid phase” as used herein refers to a wide variety of materialsincluding solids, semi-solids, gels, films, membranes, meshes, felts,composites, particles, papers and the like typically used by those ofskill in the art to sequester molecules. The solid phase can benon-porous or porous. Suitable solid phases include those developedand/or used as solid phases in solid phase binding assays. See, e.g.,chapter 9 of Immunoassay, E. P. Dianiandis and T. K. Christopoulos eds.,Academic Press: New York, 1996, hereby incorporated by reference.Examples of suitable solid phases include membrane filters,cellulose-based papers, beads (including polymeric, latex andparamagnetic particles), glass, silicon wafers, microparticles,nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, andmultiple-well plates. See, e.g., Leon et al., Bioorg. Med. Chem. Lett.8: 2997, 1998; Kessler et al., Agnew. Chem. Int. Ed. 40: 165, 2001;Smith et al., J. Comb. Med. 1: 326, 1999; Orain et al., TetrahedronLett. 42: 515, 2001; Papanikos et al., J. Am. Chem. Soc. 123: 2176,2001; Gottschling et al., Bioorg. Med. Chem. Lett. 11: 2997, 2001.

Surfaces such as those described above may be modified to providelinkage sites, for example by bromoacetylation, silation, addition ofamino groups using nitric acid, and attachment of intermediary proteins,dendrimers and/or star polymers. This list is not meant to be limiting,and any method known to those of skill in the art may be employed.

3. Detectable Label Conjugates

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate an enzyme,fluorophore or other detectable label to the molecule under study (e.g.,using one or more analogs of the invention), which may be immobilizedfor detection by a receptor molecule that has affinity for the molecule.Alternatively, the receptor to the molecule under study (e.g., anantibody or binding fragment thereof made or selected using the analogsor conjugates of the invention) may be conjugated to an enzyme,fluorophore or other detectable label. Enzyme conjugates are among themost common conjugates used. Detectable labels may include moleculesthat are themselves detectable (e.g., fluorescent moieties,electrochemical labels, metal chelates, etc.) as well as molecules thatmay be indirectly detected by production of a detectable reactionproduct (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g, biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Particularly preferred detectable labels are fluorescent latex particlessuch as those described in U.S. Pat. Nos. 5,763,189, 6,238,931, and6,251,687; and International Publication WO95/08772, each of which ishereby incorporated by reference in its entirety. Exemplary conjugationto such particles is described hereinafter.

Use of 6-AM Analogs in Receptor Binding Assays

6-AM analogs and conjugates of the present invention may beadvantageously used in receptor binding assays. Receptor binding assaysinclude any assay in which a signal is dependent upon specific bindingof an analyte to a cognate receptor, and include immunoassays,ligand-receptor assays, and nucleic acid hybridization assays.

The presence or amount of an analyte is generally determined usingantibodies specific for each marker and detecting specific binding. Anysuitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like. Specific immunological binding of the antibody tothe marker can be detected directly or indirectly. Direct labels includefluorescent or luminescent tags, metals, dyes, radionuclides, and thelike, attached to the antibody. Indirect labels include various enzymeswell known in the art, such as alkaline phosphatase, horseradishperoxidase and the like.

Numerous methods and devices are well known to the skilled artisan forthe practice of receptor binding assays. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims. These devices andmethods can utilize detectably labeled molecules and antibody solidphases in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof an analyte of interest. One skilled in the art also recognizes thatrobotic instrumentation including but not limited to Beckman Access,Abbott AxSym, Roche ElecSys, Dade Behring Stratus systems are among theimmunoassay analyzers that are capable of performing such immunoassays.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. As described herein, preferred assays utilize an antibodyraised against an analog conjugate (wherein the antibody is coupled to asolid phase or a detectable label), and/or a 6-acetylmorphine analogconjugated to a detectable label, and/or a 6-acetylmorphine analogconjugated to a solid phase.

In its simplest form, an assay device according to the invention maycomprise a solid surface comprising receptor(s) that specifically bindone or more analytes of interest (e.g., 6-AM). For example, antibodiesmay be immobilized onto a variety of solid supports, such as magnetic orchromatographic matrix particles, the surface of an assay plate (such asmicrotiter wells), pieces of a solid substrate material or membrane(such as plastic, nylon, paper), and the like using the cross-linkers ofthe present invention. In similar fashion, an assay device may comprisea solid surface comprising one or more of the 6-AM analogs describedherein immobilized thereon.

The analysis of a plurality of analytes may be carried out separately orsimultaneously with one test sample. For separate or sequential assay ofmarkers, suitable apparatuses include clinical laboratory analyzers suchas the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), theADVIA® CENTAUR® (Bayer) immunoassay systems, the NICHOLS ADVANTAGE®(Nichols Institute) immunoassay system, etc. Preferred apparatuses orprotein chips perform simultaneous assays of a plurality of analytes ona single surface. Particularly useful physical formats comprise surfaceshaving a plurality of discrete, addressable locations for the detectionof a plurality of different analytes. Such formats include proteinmicroarrays, or “protein chips” (see, e.g., Ng and Ilag, J. Cell Mol.Med. 6: 329-340 (2002)) and certain capillary devices (see, e.g., U.S.Pat. No. 6,019,944). In these embodiments, each discrete surfacelocation may comprise antibodies to immobilize one or more analyte(s)(e.g., a marker) for detection at each location. Surfaces mayalternatively comprise one or more discrete particles (e.g.,microparticles or nanoparticles) immobilized at discrete locations of asurface, where the microparticles comprise antibodies to immobilize oneanalyte (e.g., a marker) for detection.

Preparation of 6-Acetyl Morphine (6AM) Derivatives at the 1-Position ofthe A Ring in the Morphine Scaffold

The synthetic schemes are shown below and depicted in FIGS. 2-6 . Forthe synthesis of 6-phosphinyl derivative 6, morphine sulfate may beacetylated to make diacetylmorphine, followed by iodination to yield1-Iodo-diacetylmorphine derivative 2. Heck coupling of 2 with tert-butylacrylate yields enoate 3, which may be selectively reduced usingMg⁰/MeOH to give saturated diol 4. Phosphinylation of 4, followed byremoval of the aryl Phosphinyl yielded 5, may be subsequentlydeprotected using acidic conditions to yield the 6-phosphinyl derivative6 (FIG. 2 ). For the synthesis of 6-acetamide derivative 13,hydromorphine hydrochloride may be exposed to reductive amination withbenzylamine, followed by reduction to yield 6-aminohydromorphinederivative 8. The 6-amino compound is acetylated, followed by iodinationto yield 1-iodo-6-acetamide 10. Heck coupling of 10 with tert-butylacrylate yields enoate 11, which may be reduced using Mg⁰/MeOH to givesaturated 6-acetamide 12. Acidic deprotection of 12 gives 6-acetamidederivative 13 (FIG. 3 ). For the synthesis of 6-acetyl disulfide 17,saturated diol 4 may be acetylated to give 14, followed by removal ofthe phenolic acetate with hydroxylamine to yield 15, and deprotection ofthe tert-butyl ester using acidic conditions to give carboxylic acid 16.Carboxylic acid 16 may then be coupled with cystamine to give 6-acetyldisulfide 17 (FIG. 4 ). For the synthesis of sulfonamide 21,diacetylmorphine may be N-demethylated to nor-diacetylmorphine 18,followed by formation of chloride 19. The chloride can then be displacedto yield thioacetate 20, which is then deprotected to give sulfonamide21 (FIG. 5 ). For the synthesis of quaternary salt 22, diacetylmorphine1 may N-alkylated with 2-bromo-N-acetyl-HCTL (FIG. 6 ).

Morphine sulfate pentahydrate and Hydromorphone hydrochloride may beobtained from Spectrum Chemical Company. ¹H NMR spectra are typicallytaken in DMSO D₆ (from ampoules) or CDCl₃ at 500 MHz by NuMegaLaboratories. HPLC is typically conducted using an Agilent Model 1200machine equipped with either a Waters X-bridge (C18, 3.5 μm, 3.0×50 μm)or Fisher Thermo Hypercarb (5.0 um, 4.6×100 mm) columns. For HPLC,solvent A (95% H₂O/5% CH₃CN/0.1% TFA) and solvent B (95% CH₃CN/5%H₂O/0.1% TFA) may be used as described herein. HPLC runs may be either 6or 15 minutes long. For the 6 minute run: 0 minutes, 5% B, 0-5 minutes,gradient to 100% B, 5-6 minutes, gradient to 5% B; for the 15 minuterun: 0 minutes 0% B, 0-12 minutes, gradient to 100% B, 12-14 minutes100% B, 14-15 minutes, gradient to 0% B. LC/MS may be conducted using aWaters model e2795 series LC equipped with a model 2996 photodiode arraydetector, a series 3100 MS and a Waters X-Bridge-C18 column, 3.5 um,2.1×50 mm. For LC/MS, solvent A (95% H₂O/5% CH₃CN/0.1% Formic Acid) andsolvent B (95% CH₃CN/5% H₂O/0.1% Formic Acid) may be used as describedherein. HPLC runs may be 5 minutes: 0 minutes 0% B, 0-3.5 minutes,gradient to 100% B, 3.5-4.8 minutes 100% B, 4.8 to 4.9 minutes gradientto 0% B, 5.0 minutes, 0% B.

Diacetylmorphine (1): Morphine sulfate pentahydrate (1 g/1.32 mmolmorphine sulfate pentahydrate/2.64 mmol morphine) is suspended in CH₂Cl₂(10 mL) followed by the addition of NEt₃ (2.0 mL/14 mmol), pyridine (3mL) and acetic anhydride (2.4 mL/25.4 mmol). The resulting suspension isstirred at room temperature for one hour, during which time all morphinesulfate went into solution. The solution is then stirred for 14 hours atroom temperature. After this time period, additional acetic anhydride(200 μL/2.1 mmol) is added, and the solution is heated to 40° C. for 6hours. The solution is then cooled to room temperature, MeOH (7 mL) isadded, and the resulting solution stirred at room temperature for onehour before removal of the solvents under reduced pressure. Theremaining residue is partitioned in a separatory funnel between EtOAc(90 mL) and saturated NaHCO₃ (45 mL), and the biphasic mixture shakenuntil a minimum amount of gas is discharged. The organic phase is washedwith saturated NaHCO₃ (20 mL) and brine (20 mL) and dried with MgSO₄.The solvents are evaporated, and the resulting light brown residueplaced under high vacuum overnight to afford diacetylmorphine (905mg/70% yield). ¹H NMR (500 MHz, DMSO D₆) δ 6.77 (d, J=8.5 Hz, 1H), 6.63(d, J=8.0 Hz, 1H), 5.57 (m, 1H), 5.48 (m, 1H), 5.11 (m, 1H), 5.08 (m,1H); LC/MS 370 (M+H⁺).

1-Iododiacetylmorphine (2): N-Iodosuccinimide (NIS) (427 mg/1.9 mmol) isadded in one portion to a solution of 1 (460 mg/1.25 mmol) in 0.05 MH₂SO₄ (15 mL), and the resulting solution is stirred at room temperaturefor three hours before the addition of NIS (93 mg/0.4 mmol) in oneportion. The reaction is then stirred at room temperature for threehours, after which time LC/MS indicated the reaction is complete. Thereaction is then transferred to a separatory funnel containing 30 mL ofEtOAc and the reaction vessel is washed well with EtOAc. SaturatedNaHCO3 (20 mL) is then added and the separatory funnel is shaken. Thelayers are separated, and the aqueous layer is extracted with EtOAc(2×15 mL). The combined organics are washed with 2% sodium bisulfite(2×10 mL) and brine (1×10 ml), dried with MgSO₄, and the solventsremoved under reduced pressure. The crude product is purified by ISCO(24 g column, 0-10% MeOH in CH₂Cl₂) to afford the pure product as ayellow solid (618 mg/94% yield). ¹H NMR (500 MHz, DMSO D₆) δ 7.27 (s,1H), 5.53 (app. q, 2H), 5.14 (m, 1H), 5.06 (d, J=6.5 Hz, 1H); LC/MS 496(M+H⁺).

Anhydrous DMF (25 mL) is added to a vial containing 2 (1.19 g/2.4 mmol),and the solution is sparged with argon for 5 minutes, followed by theaddition of bis(triphenylphosphine)palladium(II) dichloride(Pd(PPh₃)₂Cl₂) (0.17 g/0.24 mmol), tert-butyl acrylate (1.7 mL/11.7mmol) and NEt₃ (1.3 mL/9.4 mmol). The resulting solution is heated to90° C. for 6 hours, then cooled to room temperature. EtOAc (50 mL) isadded, and the solution is transferred to a separatory funnel. Theorganic layer is washed with saturated aq NaHCO₃ (1×15 mL), and theaqueous layer is back extracted with EtOAc (2×15 mL). The combinedorganics are washed with brine (1×15 mL), dried with MgSO₄ and thesolvent removed under reduced pressure. The crude product is purified byISCO (24 g column, 0-10% MeOH in CH₂Cl₂) to afford enoate 3 as a yellowsolid (795 mg/67% yield). ¹H NMR (500 MHz, DMSO D₆) δ 7.62 (d, J=16 Hz,1H), 7.35 (s, 1H), 6.27 (d, J=16 Hz, 1H), 5.52 (app. q, 2H), 5.14 (m,1H), 5.10 (d, J=7 Hz, 1H), 1.47 (s, 9H); LC/MS 496 (M+H⁺).

Enoate 3 (828 mg/1.67 mmol) is dissolved in MeOH (12 mL, Sigma-Aldrich,anhydrous), followed by the addition of magnesium turnings (280 mg/11.5mmol) and the resulting solution is stirred at room temperature for 2hours, after which time all Mg had dissolved. Additional Mg turnings areadded (50 mg/2.1 mmol), and the reaction is stirred for 2 hours. Thesolvent is then removed under reduced pressure to yield a dark brownsolid, which is dissolved in 10 mL of CHCl₃ (bath sonication isnecessary to dissolve), and the solution is transferred to a 500 mLseparatory funnel. The reaction vial is washed with CHCl₃ (3×10 mL), and20 mL of CHCl₃ is added to the separatory funnel, followed by theaddition of 15 mL of brine. Upon the addition of brine, an emulsion isformed. An additional 30 mL of CHCl₃ is added to the funnel, and thesuspension is separated by draining the organic phase into a 1 LErlenmeyer flask. The remaining aqueous layer is extracted with CHCl₃(6×35 mL), and the combined organic phases are dried overnight bystirring with 37 g of sodium sulfate. After overnight stirring, theorganic phase is cloudy. The solution is filtered over celite. Thecelite is washed with CHCl₃ (3×40 mL), and the solvents are evaporatedto obtain 4 as an amorphous solid (230 mg/33% yield) that is usedwithout further purification in the next step. LC/MS 414 (M+H⁺).

Dimethylphosphinyl chloride is added in one portion to an oven dried 250mL round bottom flask, followed by the addition of pyridine (anhydrous,5 mL), and the resulting solution is cooled to 0° C. in an ice bath for30 minutes before the addition of tetrazole (16 mL of a 3% by masssolution in CH₃CN) in one portion. The resulting solution is stirred at0° C. for 10 minutes before the addition of a solution of diol (crude Mgreduction material 4 was) in pyridine (anhydrous, 5 mL) at the sametemperature. The solution is stirred at 0° C. for 10 minutes, followedby removal of the ice bath and allowed to warm to room temperature fortwo hours. After this time period, LC MS indicated the reaction iscomplete, only the mass of the diphosphinyl product is observed.Pyridine solvent is then removed under reduced pressure (residualpyridine is present). After removal of most of the pyridine, 30 mL ofsaturated NaHCO₃ is added, followed by 15 mL of MeOH. The resultingsolution is stirred at room temperature for 48 hours. The solution istransferred to a 250 mL separatory funnel, and the reaction flask iswashed with CH₂Cl₂ (2×15 mL). 20 mL of CH₂Cl₂ is added to the separatoryfunnel, followed by 10 mL of brine. The funnel is gently shaken, and theorganic layer is separated. The aqueous layer is extracted with CH₂Cl₂(3×20 mL), the combined organic layers are dried with MgSO₄ and thesolvents are removed under reduced pressure, The product is purified byISCO using a 24 g silica column (100% CH₂Cl₂ to 80% CH₂Cl₂:20%CH₂Cl₂:MeOH:concentrated NH₄OH (8:2:0.001) to afford 5 (176 mg/65% fromcrude 4). ¹H NMR (500 MHz, DMSO D₆) δ 8.81 (s, 1H), 6.32 (s, 1H), 5.55(d, J=10 Hz, 1H), 5.38 (d, J=10 Hz, 1H), 4.83 (m, 1H), 4.77 (d, J=5 Hz,1H), 1.55-1.44 (dd, J=15, 40 Hz, 6H), 1.37 (s, 9H); LC/MS 491 (M+H⁺).

Tert-butyl ester 5 (171 mg/0.35 mmol) is dissolved in CH₂Cl₂ (3 mL)followed by the addition of TFA:CH₂Cl₂ (3 mL:1 mL). The resultingsolution is stirred at room temperature for 2 hours, followed by removalof the solvents are removed under reduced pressure. The residue is thenplaced under high vacuum for 2 hours. After high vacuum, 1.5 mL ofCH₂Cl₂ is added, followed by the addition of HCl in ether (450 uL). Thesolvents are evaporated, and the resulting solid is evaporated withCH₂Cl₂ (1×3 mL) and CH₃CN (2×3 mL), then placed under high vacuumovernight to give 6 as an off-white solid (159 mg/99% yield). ¹H NMR(500 MHz, DMSO D₆) δ 9.10 (s, 1H), 6.44 (s, 1H), 5.69 (d, J=10 Hz, 1H),5.42 (d, J=10 Hz, 1H), 4.95 (d, J=10 Hz, 1H), 4.86 (m, 1H), 1.57-1.46(dd, J=15, 40 Hz, 6H); ³¹P NMR (125 MHz, CD₃OD) δ 60.47; LC/MS 434 (M+H⁺of free base).

To an oven dried flask equipped with a magnetic stir bar is addedhydromorphone HCl (469 mg/1.5 mmol) followed by suspending in1,2-dichloroethane (anhydrous, 12 mL). To the resulting suspension isadded benzylamine (192 μL/1.8 mmol) and sodium triacetoxyborohydride(592 mg/2.8 mmol). The resulting suspension is stirred overnight underargon at room temperature. The suspension is then transferred to aseparatory funnel, and the reaction vial is washed with CH₂Cl₂ (3×10mL). Saturated NaHCO₃ (10 mL) is added to the separatory funnel, and thecontents are shaken. The layers are separated and the aqueous layer isextracted with CH₂Cl₂ (3×10 mL). The combined organics are washed withbrine (1×5 mL), then dried with MgSO₄. The MgSO₄ is removed byfiltration, and the solvents are removed under reduced pressure to givethe crude product which is purified by ISCO (12 g column, 0-10% MeOH inCH₂Cl₂) to give 7 (484 mg/88%). ¹H NMR (500 MHz, DMSO D₆) δ 8.8 (s, 1H),6.54 (d, J=7.5 Hz), 6.44 (d, J=8.0 Hz, 1H), 4.67 (d, J=4.1 Hz, 1H), 3.78(m, 2H); LC/MS 377 (M+H⁺).

Benzyl protected amine 8 (508 mg/1.35 mmol) is dissolved in methanol (10mL) followed by degassing by sparging with argon for 10 minutes. Pd/C(102 mg) and ammonium formate (483 mg/7.7 mmol) are then added, and theresulting solution is stirred at 70° C. for 1.5 hours. The solution isthen filtered over Celite and the solvents are removed under reducedpressure. The resulting crude product is placed under high vacuumovernight. The crude product is analyzed and used without furtherpurification in the next step. ¹H NMR (500 MHz, CDCl₃) δ 6.67 (d, J=8.0Hz, 1H), 6.53 (d, J=8 Hz, 1H), 4.63 (d, J=4.0 Hz, 1H); LC/MS 287 (M+H⁺).

Amine 8 is dissolved in CH₂Cl₂ (anhydrous, 20 mL) under argon followedby the addition of acetic anhydride (433 μL/4.58 mmol) and NEt₃ (979μL/7.0 mmol) at room temperature. The resulting solution is stirredovernight at room temperature under argon. After this time period, thereaction is transferred to a separatory funnel, and the reaction flaskis washed with CH₂Cl₂ (3×10 mL). Additional CH₂Cl₂ (25 mL) is added tothe separatory funnel, followed by the addition of 5% aqueous NaHCO₃ (15mL). The funnel is shaken, and the layers are separated. The aqueouslayer is extracted with CH₂Cl₂ (2×25 mL). The combined organic layersare then washed with water and brine (1×10 mL each) and dried withMgSO₄. The organic phase is then filtered and the solvents are removedunder reduced pressure. The resulting solid is purified by ISCO (0 to10% MeOH containing 0.1% concentrated NH₄OH, 24 g column) to give thepure product as a white solid (595 mg/80%). ¹H NMR (500 MHz, DMSO D₆) δ7.30 (d, J=7.5 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.67 (d, J=8.0 Hz, 1H),4.63 (d, J=4.0 Hz, 1H), 3.94 (m, 1H); LC/MS 371 (M+H⁺).

6-Acetamide 9 is dissolved in 100 mM aqueous TFA (35 mL), followed bythe addition of NIS (230 mg/1.02 mmol) in one portion, and the resultingsolution is stirred at room temperature for two hours. After this timeperiod, additional NIS (50 mg/0.22 mmol) is added, and the solution isstirred at room temperature for two hours. The reaction is thentransferred to a separatory funnel, and of CH₂Cl₂ (50 mL) is added,followed by 5% aq. NaHCO₃ (15 mL). The funnel is shaken, and the layersare separated. The aqueous layer is then extracted with CH₂Cl₂ (3×25mL), and the combined organics are washed with 2% sodium bisulfite (2×10mL). The organic layer is dried with MgSO₄, then filtered and thesolvents removed under reduced pressure to yield the crude product. Thecrude product is purified by ISCO (24 g column, 0-10% MeOH in CH₂Cl₂) togive 10 as a yellow solid (396 mg/81% yield). ¹H NMR (500 MHz, DMSO D₆)δ 7.39 (d, J=7.5 Hz, 1H), 7.35 (s, 1H), 4.66 (d, J=4.0 Hz, 1H), 3.97 (m,1H); LC/MS 497 (M+H⁺).

DMF (anhydrous, 11 mL) is added to a vial containing iodide 10 (380mg/0.76 mmol), and the solution is sparged with argon for 5 minutesbefore the addition of of bis(triphenylphosphine)palladium(II)dichloride (Pd(PPh₃)₂Cl₂) (54 mg/0.08 mmol), tert-butyl-acrylate (0.53mL/3.65 mmol) and NEt₃ (0.42 mL/3.0 mmol). The resulting solution isheated to 90° C. for 6 hours, then cooled to room temperature. Thereaction is then transferred to a separatory funnel and the reactionvial washed with CHCl₃ (10 mL). Additional CHCl₃ (20 mL) is added to theseparatory funnel, followed by 5% NaHCO₃ (10 mL). The funnel is shakenand the layers are separated. The aqueous phase is extracted with CHCl₃(2×15 mL), and the combined organics are washed with brine (1×10 mL)before drying with MgSO₄, filtration, and removal of the solvent underreduced pressure. The crude product is purified by ISCO (24 g column, 0to 10% MeOH in CH₂Cl₂) to give 11 as an orange foam (357 mg/92%). ¹H NMR(500 MHz, DMSO D₆) δ 7.67 (d, J=15.0 Hz, 1H), 7.45 (s, 1H), 7.40 (d, J=5Hz, 1H), 6.30 (d, J=15.0 Hz, 1H), 4.70 (d, J=5 Hz, 1H), 3.98 (m, 1H),1.47 (s, 9H); LC/MS 498 (M+H⁺).

Enoate 11 (217 mg/0.44 mmol) is dissolved in MeOH (11 mL, anhydrous) ina 40 mL Thomson vial under argon, followed by the addition of Mgturnings (108 mg/4.44 mmol, oven dried at 130° C. for 2 hours, thenallowed to cool in a dessicator) in one portion. The resulting solutionis stirred under argon at room temperature for five hours, followed bythe addition of Mg turnings (20 mg/0.08 mmol), and then stirred at roomtemperature for two hours. After this time period, the solvent isremoved under reduced pressure, followed by the addition of CHCl₃ (10mL) and brine (10 mL). The resulting emulsion is filtered over sand,first by washing with CHCl₃ (50 mL), then with CHCl₃:MeOH (6:4, 200 mL).The filtrate is placed into a separatory funnel, and the aqueous andorganic phases are separated. The aqueous phase is extracted with CHCl₃(2×15 mL), and the combined organics are dried with MgSO₄, filtered, andthe solvents removed under reduced pressure. The crude product ispurified by ISCO (0-10% MeOH+0.1% concentrated NH₄OH in CH₂Cl₂) to give12. ¹H NMR (500 MHz, DMSO D₆) δ 8.95 (s, 1H), 7.50 (d, J=7.5 Hz, 1H),6.50 (s, 1H), 4.57 (m, 1H), 3.94 (m, 1H), 1.36 (s, 9H); LC/MS 457(M+H⁺).

CH₂Cl₂ (6 mL) is added to ester 12 (200 mg/0.44 mmol). The resultingsuspension is sonicated, followed by the addition of a solution ofTFA:CH₂Cl₂ (3 mL:1.5 mL), and the suspension became homogeneous followedby becoming cloudy after 5 minutes. The cloudy solution is then stirredat room temperature for 1.5 hours. The solvents are then removed underreduced pressure and the residue is placed under high vacuum for fourhours. CH₂Cl₂ (2 mL) is added to the residue, followed by the additionof HCl in Ether (1 M solution, 550 uL). The solvents are removed underreduced pressure, and the residue is evaporated with CH₂Cl₂ (2×5 mL).The product 13 is analyzed and used without further purification. ¹H NMR(500 MHz, DMSO D₆) δ 9.12 (s, 1H), 7.52 (d, J=7.5 Hz, 1H), 6.55 (s, 1H),4.63 (d, J=4.0 Hz, 1H), 3.94 (m, 1H); LC/MS 401 (M+H⁺ of free base).

To a solution of crude diol 4 (13 mg/0.031 mmol) in CH₂Cl₂ (2 mL,anhydrous) is added acetic anhydride (21 μL/0.21 mmol), NEt₃ (17 μL/0.12mmol) and DMAP (1 prill), and the resulting solution is stirredovernight at room temperature. The solution is then poured into EtOAc(10 mL), and the organic phase is washed with saturated NaHCO₃ and brine(1×5 mL each). The organic phase is dried with MgSO₄, filtered, and thesolvents removed under reduced pressure. The crude product is purifiedby preparative TLC (9:1 CHCl₃:MeOH) to give 14 as an amorphous solid (4mg/20% yield). ¹H NMR (500 MHz, DMSO D₆) δ 6.58 (s, 1H), 5.49 (app. q,2H), 5.09 (m, 1H), 4.99 (d, J=6.5 Hz, 1H), 1.35 (s, 9H); LC/MS 498(M+H⁺).

Diacetate 14 (75 mg/0.17 mmol) is added to a solution of 50 mM NaPi, pH7.5 (2 mL):MeOH (2 mL) followed by the addition of hydroxylamine (35mg/0.50 mmol) in one portion. The resulting solution is stirred at roomtemperature for four hours, followed by removal of MeOH under reducedpressure. The remaining aqueous solution is extracted with EtOAc (3×8mL), the combined organics are washed with brine (1×5 mL), dried withMgSO₄, and the solvent removed under reduced pressure. The resultingresidue is purified by preparative TLC (9:1 CHCl₃:MeOH) to give 15 as awhite solid (67 mg/85%). ¹H NMR (500 MHz, DMSO D₆) δ 9.01 (s, 1H), 6.38(s, 1H), 5.59 (d, J=9 Hz, 1H), 5.46 (d, J=10 Hz, 1H), 5.12 (m, 1H), 4.98(d, J=5 Hz, 1H); LC/MS 456 (M+H⁺).

6-Acetyl starting material (58 mg/0.13 mmol) is dissolved in 1.5 mL ofCH₂Cl₂ followed by the addition of a premixed solution of 1.5 mL ofCH₂Cl₂:1.5 mL trifluoroacetic acid (TFA). The resulting solution isstirred at room temperature for one hour. After this time period thesolvents are removed under reduced pressure and the remaining residue isplaced under high vacuum overnight. The product 16 is used withoutfurther purification in the next step (Crude yield: 75 mg/120%). LC/MS400 (M+H⁺ of free base).

To a solution of carboxylic acid 16 (8 mg/0.020 mmol) in CH₂Cl₂:DMF (2mL:0.8 mL) is added cystamine HCl (2.3 mg/0.01 mmol), HATU (10 mg/0.024mmol) and DIPEA (14 μL/0.08 mmol). The mixture is bath sonicated untilall solids are in solution and then stirred at room temperatureovernight. After this time period, solvents are removed under reducedpressure, and the resulting residue is then placed under high vacuum for4 hours to remove residual DMF. The resulting residue is purified bypreparative TLC (iPrOH:NH₄OH:H₂O 10:2:1) to give 17 as a white solid (8mg/86% yield). ¹H NMR (500 MHz, DMSO D₆) δ 9.10 (s, 1H), 8.03 (t, J=5Hz, 1H), 6.40 (s, 1H), 5.64 (d, J=10 Hz, 1H), 5.47 (d, J=9.5 Hz, 1H),5.15 (m, 1H), 5.03 (d, J=6 Hz, 1H), 2.68 (t, J=9.5 Hz, 2H), 2.41 (t, J=7Hz, 2H); LC/MS 913 (M−H).

Diacetylmorphine (1, 242 mg/0.66 mmol) is added to an oven dried flaskequipped with a magnetic stir bar followed by the addition of toluene (6mL), potassium carbonate (182 mg/1.84 mmol) and trichloroethylchloroformate (0.36 mL/2.64 mmol) at room temperature. The resultingsuspension is then refluxed at 120° C. for 20 hours. After this timeperiod, LC/MS indicated starting material remained. The solution is thencooled to room temp, additional trichloroethyl chloroformate is added(0.20 mL/1.47 mmol), and the solution is refluxed at 120° C. for 8hours. After this time period the reaction is complete, as monitored byLC/MS. Potassium carbonate is then removed by filtration and toluene isremoved under reduced pressure. To the resulting residue is added THF(0.20 mL) and 90% acetic acid (0.105 mL), and the suspension is stirredat room temperature for 6 hours, after which time the reaction iscomplete as monitored by LC/MS. The solution is separated from themajority of the solid Zn by pipette, and filtered through coarse filterpaper into a separatory funnel. The filter paper is washed withisopropanol (5 mL), CHCl₃ (40 mL) and H₂O (20 mL). The aqueous layer issaturated with NaCl, followed by the addition of 50% aqueous NH₂OHdropwise until the pH of the solution reached approximately 7 (pHpaper). During this time the funnel is shaken periodically to promoteextraction of 18 into the organic phase. CHCl₃ is then separated, and anew portion of CHCl₃ (20 mL) is added before the pH of the solution isadjusted to approximately 9 using 50% aqueous NH₂OH. The separatoryfunnel is shaken during the pH adjustment periodically. The organiclayer is separated, and the resulting aqueous solution is extracted withCHCl₃ (3×15 mL). The combined organic layers are dried with MgSO₄, andthe solvents removed under reduced pressure to yield the crude productas a yellow oil (233 mg/100% crude yield); LC/MS 356 (M+H⁺). The crudeproduct is used in the next step without further purification.

Nordiacetylmorphine (18) (65 mg/0.18 mmol) is dissolved in anhydrousCH₂Cl₂ (3 mL), and the solution is cooled to 0° C., followed by theaddition of DIPEA (0.063 mL/0.36 mmol) and 3-chloropropylsulfonylchloride (0.024 mL/0.20 mmol) at 0° C. The reaction is stirred at 0° C.for one hour, then allowed to warm to room temperature with stirringovernight. H₂O (4 mL) and EtOAc (10 mL) are then added. The layers areseparated, and the aqueous layer is extracted with EtOAc (2×5 mL). Thecombined organic layers are washed with H₂O (1×5 mL) and brine (1×5 mL),dried over MgSO₄ and the solvent removed under reduced pressure. Thecrude product is purified by preparative TLC (1:2 Hexanes:EtOAc) to give19 as an amorphous solid (40 mg/44% yield). ¹H NMR (500 MHz, CDCl₃) δ6.82 (d, J=8.2 Hz, 1H), 6.62 (d, J=8.2 Hz, 1H), 5.71 (d, J=11.4 Hz, 1H),5.42 (d, J=10.3 Hz, 1H), 3.71 (t, J=5.9 Hz, 2H), 2.28 (s, 3H), 2.14 (s,3H); LC/MS 495 (M−H).

Chloride 19 (128 mg/0.26 mmol) is dissolved in anhydrous DMF (4 mL)followed by the addition of potassium thioacetate (148 mg/1.30 mmol),and the solution is heated to 90° C. for 5 hours. The reaction is cooledto room temperature, EtOAc (15 mL) is added, and the solution istransferred to a separatory funnel. The organic phase is washed with H₂O(1×5 mL) and brine (2×5 mL), dried with MgSO₄ and the solvents removedunder reduced pressure to yield the crude product which is purified byISCO (20:1 Hexanes:EtOAc to 5:1 Hexanes:EtOAc) to give thioacetate 20 asan amorphous solid (85 mg/62% yield). ¹H NMR (500 MHz, CDCl₃) δ 6.81 (d,J=9.6 Hz, 1H), 6.61 (d, J=8.3 Hz, 1H), 5.71 (d, J=10.0 Hz, 1H), 5.42 (d,J=10.1 Hz, 1H), 3.06 (t, J=7.1 Hz, 2H), 2.36 (s, 3H), 2.28 (s, 3H);LC/MS 534 (M−H).

Thioacetate 20 (29 mg/0.054 mmol) is dissolved in CH₃OH/THF (2 mL:0.6mL), followed by the addition of 100 mM sodium phosphate buffer (pH7.0). 15 mg of hydroxylamine hydrochloride is then added, and theresulting solution is stirred overnight at room temperature. Thecontents of the vial are transferred to a separatory funnel, EtOAc (10mL) and water (5 mL) are added, and the funnel is shaken. The layers areseparated, and the aqueous layer is extracted with EtOAc (1×5 mL). Thecombined organic layers are washed with brine (1×5 mL), dried with MgSO₄and the solvent removed under reduced pressure. The crude product ispurified by preparative HPLC to give the pure product as the disulfidederivative of 21 (LC/MS) (2.6 mg/10% yield). Free thiol 21 is obtainedby dissolving the disulfide in DMSO:sodium phosphate buffer (pH 7.5)(400 uL DMSO:440 uL 50 mM NaPi), followed by the addition of TCEP (2.2mg/0.008 mmol) in one portion. The resulting solution is stirred at roomtemperature for 30 minutes to yield 21. LC/MS 452 (M−H).

Diacetylmorphine 1 (10 mg, 0.03 mmol) is dissolved in acetonitrile (1mL). To the mixture is added 2-bromo-N-acetyl-HCTL (16 mg, 0.067 mmol).The reaction mixture is then heated at 60° C. for overnight. Afterovernight, the reaction mixture is cooled down to room temperature. Themixture is then purified using silica TLC plate, eluted with 10%MeOH/DCM to afford 3.6 mg (22%) of 22 as a white powder. ¹H NMR(DMSO-d6) δ 9.14 (1H, d), 6.88 (1H, d), 6.71 (1H, d), 5.65 (1H, d), 5.53(1H, d); LC/MS 528 (M+).

Conjugation Chemistries: Preparation of Conjugates of 6-Acetyl Morphine(6AM)

A 6-AM derivative 23 having a linking group containing a sulfhydrylgroup reactive with maleimide or haloacetyl or haloacetamido moiety:

may be used to prepare conjugates to a latex solid phase and to KLH asdescribed below.

Bovine serum albumin (“BSA”) and polystyrene latex particles(Interfacial Dynamics) are incubated at 25° C. for 30 minutes at 1-10 mgBSA per mL of latex slurry at 1-10% solids in 25 mM citrate buffer, pHapproximately 4. The solution is then brought to approximately neutralpH with 150 mM potassium phosphate/30 mM potassium borate, and incubatedfor an additional 2 hours at 25° C. The suspension is washed three timesby resuspension in 50 mM potassium phosphate/10 mM potassium borate/150mM sodium chloride at approximately neutral pH followed bycentrifugation.

An N-hydroxysuccinimide/maleimide bifunctional poly(ethylene glycol)crosslinker as described in U.S. Pat. No. 6,887,952 is added at 5-500mg/mL in deionized water to the BSA-latex particles at 1-10% solids. Thecrosslinker is incubated with the BSA-latex particles at roomtemperature for 2 hours. Excess crosslinker is removed by centrifugationand resuspension in PBS of the now maleimide-functionalized BSA-latexparticles.

The derivative (4-8 mg) is dissolved in 0.8 mL DMF-water solution (70:30v/v) and 200 μL of 1 M KOH, and is incubated for 10 minutes at roomtemperature. Then the excess of the base is neutralized with aphosphate/hydrochloric acid buffer to pH 7. Maleimide-functionalizedBSA-latex particles are added to the solution containing the 6-AMderivative in the presence of 0.1 mM EDTA, and the mixture is incubatedat room temperature overnight. KOH is added to maintain the pH at about7.0. The reaction is stopped in two steps. First by addition of 0.2 mMβ-mercaptoethanol and incubation for 30 at room temperature and then byaddition of 6 mM N-(hydroxyethyl)maleimide and additional incubation for30 minutes at room temperature. The 6-AM derivative-conjugated latexparticles are purified by centrifugation and resuspension in PBS.

Keyhole Limpet Hemocyanin (KLH, Calbiochem #374817, 50 mg/mL inglycerol) is passed through a 40 mL GH25 column equilibrated in 0.1Mpotassium phosphate, 0.1M borate, 0.15M sodium chloride buffer, pH 7.5to remove glycerol. A 1.5-fold molar excess of N-ethylmaleimide isadded, and the mixture incubated 30 minutes at room temperature. A200-fold molar excess of sulfo-SMCC (Pierce #22322) from a 50 mM stockin distilled water is added while vortexing. Vortexing is continued foranother 30 seconds, followed by incubation for 10 minutes at roomtemperature. A 100-fold molar excess of SMCC (Pierce #22360) from an 80mM stock in acetonitrile is added while vortexing. 1M KOH is added tomaintain a pH of between 7.2 and 7.4. The mixture is stirred at roomtemperature for 90 minutes. After 90 minutes incubation, KLH-SMCC ispurified by gel filtration using a GH25 column equilibrated in 0.1Mpotassium phosphate, 0.02M borate, 0.15M sodium chloride buffer, pH 7.0.

The 6-AM derivative (4-8 mg) is dissolved in 0.8 mL DMF-water solution(70:30 v/v) and 200 μL of 1 M KOH, and is incubated for 10 minutes atroom temperature. The excess of the base is neutralized with aphosphate/hydrochloric acid buffer and pH brought to 7. Then, a 2-foldmolar excess of derivative (based on the concentration of SMCC in aparticular batch of KLH-SMCC) is added to KLH-SMCC, and the mixturestirred for 90 minutes at room temperature. Conjugates are purified byexhaustive dialysis in PBS.

Preparation of Antibodies Against 6-Acetyl Morphine (6AM)

Following immunization with the KLH-conjugated derivative, phage displayantibody libraries may be constructed and enriched usingbiotin-conjugated 24 and magnetic streptavidin latex as generallydescribed in U.S. Pat. No. 6,057,098. The antibody phage library isselected with 24, transferred into a plasmid expression vector andelectro-porated into bacterial cells. Simultaneous negative selection isperformed with 25, 26, and 27 to select against antibodies binding toundesired epitopes.

The bacterial cells from each antibody library are streaked on agar togenerate colonies. The colonies, coding for monoclonal antibodies, areused to inoculate culture medium in individual wells in 96-well plates.The liquid cultures are grown overnight and used to generate frozen cellstocks. The frozen cell stocks are used to generate duplicate 96-wellplate cultures, followed by expression and purification of themonoclonal antibodies in soluble form in microgram quantities. Acompetitive assay for 6-AM developed with a selected antibody exhibitedno crossreactivity with morphine, morphine-3-glucuronide, ormorphine-6-glucuronide at clinically relevant concentrations.

Evaluation of Cross-Reactivity of Antibodies Against 6-Acetyl Morphine(6AM) with Other Heroin Metabolites

Cross reactivity to heroin metabolites and other common structurallyrelated opiates may be evaluated using the antibodies described herein.Immunoassays are constructed using the antibodies of Example 3,configured to operate in a competitive mode immunoassay format, in whichthe analogue compounds of the invention are compared with other relatedcompounds for cross reaction against 6-acetylmorphine. Labeled6-acetylmorphine conjugates are prepared for use as the detectablespecies. Aliquots of labeled 6-acetylmorphine at 10 ng/mL are incubatedin the presence of competing compound with the antibody of theinvention, and the level of interaction of the competitor compounddetermined as a reduction in measured signal compared with the situationwhere only 6-acetylmorphine is present. The results are provided in theTable 1. The data demonstrate the high specificity of the antibody for6-acetylmorphine. With many of the competing species being applied at anexcess of 100,000 to 1 over 6-acetylmorphine; there is no detectablecross reaction; and with 6-acetylcodeine at a 30:1 excess over6-acetylmorphone there is only a 3% cross reaction. The data clearlyindicate the specificity of the antibodies of the invention for6-acetylmorphine.

EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1: 6-Acetyl Morphine (6AM) Derivatives at the 1-Position of theA Ring in the Morphine Scaffold

The synthetic schemes are shown below and depicted in FIGS. 2-6 . Forthe synthesis of 6-phosphinyl derivative 6, morphine sulfate wasacetylated to make diacetylmorphine, followed by iodination to yield1-Iodo-diacetylmorphine derivative 2. Heck coupling of 2 with tert-butylacrylate yielded enoate 3, which was selectively reduced using MgO/MeOHto give saturated diol 4. Phosphinylation of 4, followed by removal ofthe aryl Phosphinyl yielded 5, which was deprotected using acidicconditions to yield the 6-phosphinyl derivative 6 (FIG. 2 ). For thesynthesis of 6-acetamide derivative 13, hydromorphine hydrochloride wasexposed to reductive amination with benzylamine, followed by reductionto yield 6-aminohydromorphine derivative 8. The 6-amino compound wasacetylated, followed by iodination to yield 1-iodo-6-acetamide 10. Heckcoupling of 10 with tert-butyl acrylate yielded enoate 11, which wasreduced using MgO/MeOH to give saturated 6-acetamide 12. Acidicdeprotection of 12 gave 6-acetamide derivative 13 (FIG. 3 ). For thesynthesis of 6-acetyl disulfide 17, saturated diol 4 was acetylated togive 14, followed by removal of the phenolic acetate with hydroxylamineto yield 15, and deprotection of the tert-butyl ester using acidicconditions to give carboxylic acid 16. Carboxylic acid 16 was thencoupled with cystamine to give 6-acetyl disulfide 17 (FIG. 4 ). For thesynthesis of sulfonamide 21, diacetylmorphine was N-demethylated tonor-diacetylmorphine 18, followed by formation of chloride 19. Thechloride was displaced to yield thioacetate 20, which was deprotected togive sulfonamide 21 (FIG. 5 ). For the synthesis of quaternary salt 22,diacetylmorphine 1 was N-alkylated with 2-bromo-N-acetyl-HCTL (FIG. 6 ).

General Methods

All starting materials and solvents were obtained from commercialvendors unless otherwise noted. Morphine sulfate pentahydrate andHydromorphone hydrochloride were obtained from Spectrum ChemicalCompany. ¹H NMR spectra were taken in DMSO D₆ (from ampoules) or CDCl₃at 500 MHz by NuMega Laboratories. HPLC was conducted using an AgilentModel 1200 machine equipped with either a Waters X-bridge (C18, 3.5 μm,3.0×50 μm) or Fisher Thermo Hypercarb (5.0 um, 4.6×100 mm) columns. ForHPLC, solvent A was 95% H₂O/5% CH₃CN/0.1% TFA, solvent B was 95%CH₃CN/5% H₂O/0.1% TFA. HPLC runs were either 6 or 15 minutes long. Forthe 6 minute run: 0 minutes, 5% B, 0-5 minutes, gradient to 100% B, 5-6minutes, gradient to 5% B; for the 15 minute run: 0 minutes 0% B, 0-12minutes, gradient to 100% B, 12-14 minutes 100% B, 14-15 minutes,gradient to 0% B. LC/MS was conducted using a Waters model e2795 seriesLC equipped with a model 2996 photodiode array detector, a series 3100MS and a Waters X-Bridge-C18 column, 3.5 um, 2.1×50 mm. For LC/MS,solvent A was 95% H₂O/5% CH₃CN/0.1% Formic Acid; solvent B was 95%CH₃CN/5% H₂O/0.1% Formic Acid. HPLC runs were 5 minutes: 0 minutes 0% B,0-3.5 minutes, gradient to 100% B, 3.5-4.8 minutes 100% B, 4.8 to 4.9minutes gradient to 0% B, 5.0 minutes, 0% B.

Synthetic Procedures

Diacetylmorphine (1): Morphine sulfate pentahydrate (1 g/1.32 mmolmorphine sulfate pentahydrate/2.64 mmol morphine) was suspended inCH₂Cl₂ (10 mL) followed by the addition of NEt₃ (2.0 mL/14 mmol),pyridine (3 mL) and acetic anhydride (2.4 mL/25.4 mmol). The resultingsuspension was stirred at room temperature for one hour, during whichtime all morphine sulfate went into solution. The solution was thenstirred for 14 hours at room temperature. After this time period,additional acetic anhydride (200 μL/2.1 mmol) was added, and thesolution was heated to 40° C. for 6 hours. The solution was then cooledto room temperature, MeOH (7 mL) was added, and the resulting solutionstirred at room temperature for one hour before removal of the solventsunder reduced pressure. The remaining residue was partitioned in aseparatory funnel between EtOAc (90 mL) and saturated NaHCO₃ (45 mL),and the biphasic mixture shaken until a minimum amount of gas wasdischarged. The organic phase was washed with saturated NaHCO₃ (20 mL)and brine (20 mL) and dried with MgSO₄. The solvents were evaporated,and the resulting light brown residue placed under high vacuum overnightto afford diacetylmorphine (905 mg/70% yield). ¹H NMR (500 MHz, DMSO D₆)δ 6.77 (d, J=8.5 Hz, 1H), 6.63 (d, J=8.0 Hz, 1H), 5.57 (m, 1H), 5.48 (m,1H), 5.11 (m, 1H), 5.08 (m, 1H); LC/MS 370 (M+H⁺).

1-Iododiacetylmorphine (2): N-Iodosuccinimide (NIS) (427 mg/1.9 mmol)was added in one portion to a solution of 1 (460 mg/1.25 mmol) in 0.05 MH₂SO₄ (15 mL), and the resulting solution was stirred at roomtemperature for three hours before the addition of NIS (93 mg/0.4 mmol)in one portion. The reaction was then stirred at room temperature forthree hours, after which time LC/MS indicated the reaction was complete.The reaction was then transferred to a separatory funnel containing 30mL of EtOAc and the reaction vessel was washed well with EtOAc.Saturated NaHCO₃ (20 mL) was then added and the separatory funnel wasshaken. The layers were separated, and the aqueous layer was extractedwith EtOAc (2×15 mL). The combined organics were washed with 2% sodiumbisulfite (2×10 mL) and brine (1×10 ml), dried with MgSO₄, and thesolvents removed under reduced pressure. The crude product was purifiedby ISCO (24 g column, 0-10% MeOH in CH₂Cl₂) to afford the pure productas a yellow solid (618 mg/94% yield). ¹H NMR (500 MHz, DMSO D₆) δ 7.27(s, 1H), 5.53 (app. q, 2H), 5.14 (m, 1H), 5.06 (d, J=6.5 Hz, 1H); LC/MS496 (M+H⁺).

Anhydrous DMF (25 mL) was added to a vial containing 2 (1.19 g/2.4mmol), and the solution was sparged with argon for 5 minutes, followedby the addition of bis(triphenylphosphine)palladium(II) dichloride(Pd(PPh₃)₂Cl₂) (0.17 g/0.24 mmol), tert-butyl acrylate (1.7 mL/11.7mmol) and NEt₃ (1.3 mL/9.4 mmol). The resulting solution was heated to90° C. for 6 hours, then cooled to room temperature. EtOAc (50 mL) wasadded, and the solution was transferred to a separatory funnel. Theorganic layer was washed with saturated aq NaHCO₃ (1×15 mL), and theaqueous layer was back extracted with EtOAc (2×15 mL). The combinedorganics were washed with brine (1×15 mL), dried with MgSO₄ and thesolvent removed under reduced pressure. The crude product was purifiedby ISCO (24 g column, 0-10% MeOH in CH₂Cl₂) to afford enoate 3 as ayellow solid (795 mg/67% yield). ¹H NMR (500 MHz, DMSO D₆) δ 7.62 (d,J=16 Hz, 1H), 7.35 (s, 1H), 6.27 (d, J=16 Hz, 1H), 5.52 (app. q, 2H),5.14 (m, 1H), 5.10 (d, J=7 Hz, 1H), 1.47 (s, 9H); LC/MS 496 (M+H⁺).

Enoate 3 (828 mg/1.67 mmol) was dissolved in MeOH (12 mL, Sigma-Aldrich,anhydrous), followed by the addition of magnesium turnings (280 mg/11.5mmol) and the resulting solution was stirred at room temperature for 2hours, after which time all Mg had dissolved. Additional Mg turningswere added (50 mg/2.1 mmol), and the reaction was stirred for 2 hours.The solvent was then removed under reduced pressure to yield a darkbrown solid, which was dissolved in 10 mL of CHCl₃ (bath sonication wasnecessary to dissolve), and the solution was transferred to a 500 mLseparatory funnel. The reaction vial was washed with CHCl₃ (3×10 mL),and 20 mL of CHCl₃ was added to the separatory funnel, followed by theaddition of 15 mL of brine. Upon the addition of brine, an emulsion wasformed. An additional 30 mL of CHCl₃ was added to the funnel, and thesuspension was separated by draining the organic phase into a 1 LErlenmeyer flask. The remaining aqueous layer was extracted with CHCl₃(6×35 mL), and the combined organic phases were dried overnight bystirring with 37 g of sodium sulfate. After overnight stirring, theorganic phase was cloudy. The solution was filtered over celite. Thecelite was washed with CHCl₃ (3×40 mL), and the solvents were evaporatedto obtain 4 as an amorphous solid (230 mg/33% yield) that was usedwithout further purification in the next step. LC/MS 414 (M+H⁺).

Dimethylphosphinyl chloride was added in one portion to an oven dried250 mL round bottom flask, followed by the addition of pyridine(anhydrous, 5 mL), and the resulting solution was cooled to 0° C. in anice bath for 30 minutes before the addition of tetrazole (16 mL of a 3%by mass solution in CH₃CN) in one portion. The resulting solution wasstirred at 0° C. for 10 minutes before the addition of a solution ofdiol (crude Mg reduction material 4 was) in pyridine (anhydrous, 5 mL)at the same temperature. The solution was stirred at 0° C. for 10minutes, followed by removal of the ice bath and allowed to warm to roomtemperature for two hours. After this time period, LC MS indicated thereaction was complete, only the mass of the diphosphinyl product wasobserved. Pyridine solvent was then removed under reduced pressure(residual pyridine was present). After removal of most of the pyridine,30 mL of saturated NaHCO₃ was added, followed by 15 mL of MeOH. Theresulting solution was stirred at room temperature for 48 hours. Thesolution was transferred to a 250 mL separatory funnel, and the reactionflask was washed with CH₂Cl₂ (2×15 mL). 20 mL of CH₂Cl₂ was added to theseparatory funnel, followed by 10 mL of brine. The funnel was gentlyshaken, and the organic layer was separated. The aqueous layer wasextracted with CH₂Cl₂ (3×20 mL), the combined organic layers were driedwith MgSO₄ and the solvents were removed under reduced pressure, Theproduct was purified by ISCO using a 24 g silica column (100% CH₂Cl₂ to80% CH₂Cl₂:20% CH₂Cl₂:MeOH:concentrated NH₄OH (8:2:0.001) to afford 5(176 mg/65% from crude 4). ¹H NMR (500 MHz, DMSO D₆) δ 8.81 (s, 1H),6.32 (s, 1H), 5.55 (d, J=10 Hz, 1H), 5.38 (d, J=10 Hz, 1H), 4.83 (m,1H), 4.77 (d, J=5 Hz, 1H), 1.55-1.44 (dd, J=15, 40 Hz, 6H), 1.37 (s,9H); LC/MS 491 (M+H⁺).

Tert-butyl ester 5 (171 mg/0.35 mmol) was dissolved in CH₂Cl₂ (3 mL)followed by the addition of TFA:CH₂Cl₂ (3 mL:1 mL). The resultingsolution was stirred at room temperature for 2 hours, followed byremoval of the solvents were removed under reduced pressure. The residuewas then placed under high vacuum for 2 hours. After high vacuum, 1.5 mLof CH₂Cl₂ was added, followed by the addition of HCl in ether (450 uL).The solvents were evaporated, and the resulting solid was evaporatedwith CH₂Cl₂ (1×3 mL) and CH₃CN (2×3 mL), then placed under high vacuumovernight to give 6 as an off-white solid (159 mg/99% yield). ¹H NMR(500 MHz, DMSO D₆) δ 9.10 (s, 1H), 6.44 (s, 1H), 5.69 (d, J=10 Hz, 1H),5.42 (d, J=10 Hz, 1H), 4.95 (d, J=10 Hz, 1H), 4.86 (m, 1H), 1.57-1.46(dd, J=15, 40 Hz, 6H); ³¹P NMR (125 MHz, CD₃OD) δ 60.47; LC/MS 434 (M+H⁺of free base).

To an oven dried flask equipped with a magnetic stir bar was addedhydromorphone HCl (469 mg/1.5 mmol) followed by suspending in1,2-dichloroethane (anhydrous, 12 mL). To the resulting suspension wasadded benzylamine (192 μL/1.8 mmol) and sodium triacetoxyborohydride(592 mg/2.8 mmol). The resulting suspension was stirred overnight underargon at room temperature. The suspension was then transferred to aseparatory funnel, and the reaction vial was washed with CH₂Cl₂ (3×10mL). Saturated NaHCO₃ (10 mL) was added to the separatory funnel, andthe contents were shaken. The layers were separated and the aqueouslayer was extracted with CH₂Cl₂ (3×10 mL). The combined organics werewashed with brine (1×5 mL), then dried with MgSO₄. The MgSO₄ was removedby filtration, and the solvents were removed under reduced pressure togive the crude product which was purified by ISCO (12 g column, 0-10%MeOH in CH₂Cl₂) to give 7 (484 mg/88%). ¹H NMR (500 MHz, DMSO D₆) δ 8.8(s, 1H), 6.54 (d, J=7.5 Hz), 6.44 (d, J=8.0 Hz, 1H), 4.67 (d, J=4.1 Hz,1H), 3.78 (m, 2H); LC/MS 377 (M+H⁺).

Benzyl protected amine 8 (508 mg/1.35 mmol) was dissolved in methanol(10 mL) followed by degassing by sparging with argon for 10 minutes.Pd/C (102 mg) and ammonium formate (483 mg/7.7 mmol) were then added,and the resulting solution was stirred at 70° C. for 1.5 hours. Thesolution was then filtered over Celite and the solvents were removedunder reduced pressure. The resulting crude product was placed underhigh vacuum overnight. The crude product was analyzed and used withoutfurther purification in the next step. ¹H NMR (500 MHz, CDCl₃) δ 6.67(d, J=8.0 Hz, 1H), 6.53 (d, J=8 Hz, 1H), 4.63 (d, J=4.0 Hz, 1H); LC/MS287 (M+H⁺).

Amine 8 was dissolved in CH₂Cl₂ (anhydrous, 20 mL) under argon followedby the addition of acetic anhydride (433 μL/4.58 mmol) and NEt₃ (979μL/7.0 mmol) at room temperature. The resulting solution was stirredovernight at room temperature under argon. After this time period, thereaction was transferred to a separatory funnel, and the reaction flaskwas washed with CH₂Cl₂ (3×10 mL). Additional CH₂Cl₂ (25 mL) was added tothe separatory funnel, followed by the addition of 5% aqueous NaHCO₃ (15mL). The funnel was shaken, and the layers were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×25 mL). The combined organic layerswere then washed with water and brine (1×10 mL each) and dried withMgSO₄. The organic phase was then filtered and the solvents were removedunder reduced pressure. The resulting solid was purified by ISCO (0 to10% MeOH containing 0.1% concentrated NH₄OH, 24 g column) to give thepure product as a white solid (595 mg/80%). ¹H NMR (500 MHz, DMSO D₆) δ7.30 (d, J=7.5 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.67 (d, J=8.0 Hz, 1H),4.63 (d, J=4.0 Hz, 1H), 3.94 (m, 1H); LC/MS 371 (M+H⁺).

6-Acetamide 9 was dissolved in 100 mM aqueous TFA (35 mL), followed bythe addition of NIS (230 mg/1.02 mmol) in one portion, and the resultingsolution was stirred at room temperature for two hours. After this timeperiod, additional NIS (50 mg/0.22 mmol) was added, and the solution wasstirred at room temperature for two hours. The reaction was thentransferred to a separatory funnel, and of CH₂Cl₂ (50 mL) was added,followed by 5% aq. NaHCO₃ (15 mL). The funnel was shaken, and the layerswere separated. The aqueous layer was then extracted with CH₂Cl₂ (3×25mL), and the combined organics were washed with 2% sodium bisulfite(2×10 mL). The organic layer was dried with MgSO₄, then filtered and thesolvents removed under reduced pressure to yield the crude product. Thecrude product was purified by ISCO (24 g column, 0-10% MeOH in CH₂Cl₂)to give 10 as a yellow solid (396 mg/81% yield). ¹H NMR (500 MHz, DMSOD₆) δ 7.39 (d, J=7.5 Hz, 1H), 7.35 (s, 1H), 4.66 (d, J=4.0 Hz, 1H), 3.97(m, 1H); LC/MS 497 (M+H⁺).

DMF (anhydrous, 11 mL) was added to a vial containing iodide 10 (380mg/0.76 mmol), and the solution was sparged with argon for 5 minutesbefore the addition of of bis(triphenylphosphine)palladium(II)dichloride (Pd(PPh₃)₂Cl₂) (54 mg/0.08 mmol), tert-butyl-acrylate (0.53mL/3.65 mmol) and NEt₃ (0.42 mL/3.0 mmol). The resulting solution washeated to 90° C. for 6 hours, then cooled to room temperature. Thereaction was then transferred to a separatory funnel and the reactionvial washed with CHCl₃ (10 mL). Additional CHCl₃ (20 mL) was added tothe separatory funnel, followed by 5% NaHCO₃ (10 mL). The funnel wasshaken and the layers were separated. The aqueous phase was extractedwith CHCl₃ (2×15 mL), and the combined organics were washed with brine(1×10 mL) before drying with MgSO₄, filtration, and removal of thesolvent under reduced pressure. The crude product was purified by ISCO(24 g column, 0 to 10% MeOH in CH₂Cl₂) to give 11 as an orange foam (357mg/92%). ¹H NMR (500 MHz, DMSO D₆) δ 7.67 (d, J=15.0 Hz, 1H), 7.45 (s,1H), 7.40 (d, J=5 Hz, 1H), 6.30 (d, J=15.0 Hz, 1H), 4.70 (d, J=5 Hz,1H), 3.98 (m, 1H), 1.47 (s, 9H); LC/MS 498 (M+H⁺).

Enoate 11 (217 mg/0.44 mmol) was dissolved in MeOH (11 mL, anhydrous) ina 40 mL Thomson vial under argon, followed by the addition of Mgturnings (108 mg/4.44 mmol, oven dried at 130° C. for 2 hours, thenallowed to cool in a dessicator) in one portion. The resulting solutionwas stirred under argon at room temperature for five hours, followed bythe addition of Mg turnings (20 mg/0.08 mmol), and then stirred at roomtemperature for two hours. After this time period, the solvent wasremoved under reduced pressure, followed by the addition of CHCl₃ (10mL) and brine (10 mL). The resulting emulsion was filtered over sand,first by washing with CHCl₃ (50 mL), then with CHCl₃:MeOH (6:4, 200 mL).The filtrate was placed into a separatory funnel, and the aqueous andorganic phases were separated. The aqueous phase was extracted withCHCl₃ (2×15 mL), and the combined organics were dried with MgSO₄,filtered, and the solvents removed under reduced pressure. The crudeproduct was purified by ISCO (0-10% MeOH+0.1% concentrated NH₄OH inCH₂Cl₂) to give 12. ¹H NMR (500 MHz, DMSO D₆) δ 8.95 (s, 1H), 7.50 (d,J=7.5 Hz, 1H), 6.50 (s, 1H), 4.57 (m, 1H), 3.94 (m, 1H), 1.36 (s, 9H);LC/MS 457 (M+H⁺).

CH₂Cl₂ (6 mL) was added to ester 12 (200 mg/0.44 mmol). The resultingsuspension was sonicated, followed by the addition of a solution ofTFA:CH₂Cl₂ (3 mL:1.5 mL), and the suspension became homogeneous followedby becoming cloudy after 5 minutes. The cloudy solution was then stirredat room temperature for 1.5 hours. The solvents were then removed underreduced pressure and the residue was placed under high vacuum for fourhours. CH₂Cl₂ (2 mL) was added to the residue, followed by the additionof HCl in Ether (1 M solution, 550 uL). The solvents were removed underreduced pressure, and the residue was evaporated with CH₂Cl₂ (2×5 mL).The product 13 was analyzed and used without further purification. ¹HNMR (500 MHz, DMSO D₆) δ 9.12 (s, 1H), 7.52 (d, J=7.5 Hz, 1H), 6.55 (s,1H), 4.63 (d, J=4.0 Hz, 1H), 3.94 (m, 1H); LC/MS 401 (M+H⁺ of freebase).

To a solution of crude diol 4 (13 mg/0.031 mmol) in CH₂Cl₂ (2 mL,anhydrous) was added acetic anhydride (21 μL/0.21 mmol), NEt₃ (17μL/0.12 mmol) and DMAP (1 prill), and the resulting solution was stirredovernight at room temperature. The solution was then poured into EtOAc(10 mL), and the organic phase was washed with saturated NaHCO₃ andbrine (1×5 mL each). The organic phase was dried with MgSO₄, filtered,and the solvents removed under reduced pressure. The crude product waspurified by preparative TLC (9:1 CHCl₃:MeOH) to give 14 as an amorphoussolid (4 mg/20% yield). ¹H NMR (500 MHz, DMSO D₆) δ 6.58 (s, 1H), 5.49(app. q, 2H), 5.09 (m, 1H), 4.99 (d, J=6.5 Hz, 1H), 1.35 (s, 9H); LC/MS498 (M+H⁺).

Diacetate 14 (75 mg/0.17 mmol) was added to a solution of 50 mM NaPi, pH7.5 (2 mL):MeOH (2 mL) followed by the addition of hydroxylamine (35mg/0.50 mmol) in one portion. The resulting solution was stirred at roomtemperature for four hours, followed by removal of MeOH under reducedpressure. The remaining aqueous solution was extracted with EtOAc (3×8mL), the combined organics were washed with brine (1×5 mL), dried withMgSO₄, and the solvent removed under reduced pressure. The resultingresidue was purified by preparative TLC (9:1 CHCl₃:MeOH) to give 15 as awhite solid (67 mg/85%). ¹H NMR (500 MHz, DMSO D₆) δ 9.01 (s, 1H), 6.38(s, 1H), 5.59 (d, J=9 Hz, 1H), 5.46 (d, J=10 Hz, 1H), 5.12 (m, 1H), 4.98(d, J=5 Hz, 1H); LC/MS 456 (M+H⁺).

6-Acetyl starting material (58 mg/0.13 mmol) was dissolved in 1.5 mL ofCH₂Cl₂ followed by the addition of a premixed solution of 1.5 mL ofCH₂Cl₂:1.5 mL trifluoroacetic acid (TFA). The resulting solution wasstirred at room temperature for one hour. After this time period thesolvents were removed under reduced pressure and the remaining residuewas placed under high vacuum overnight. The product 16 was used withoutfurther purification in the next step (Crude yield: 75 mg/120%). LC/MS400 (M+H⁺ of free base).

To a solution of carboxylic acid 16 (8 mg/0.020 mmol) in CH₂Cl₂:DMF (2mL:0.8 mL) was added cystamine HCl (2.3 mg/0.01 mmol), HATU (10 mg/0.024mmol) and DIPEA (14 μL/0.08 mmol). The mixture was bath sonicated untilall solids were in solution and then stirred at room temperatureovernight. After this time period, solvents were removed under reducedpressure, and the resulting residue was then placed under high vacuumfor 4 hours to remove residual DMF. The resulting residue was purifiedby preparative TLC (iPrOH:NH₄OH:H₂O 10:2:1) to give 17 as a white solid(8 mg/86% yield). ¹H NMR (500 MHz, DMSO D₆) δ 9.10 (s, 1H), 8.03 (t, J=5Hz, 1H), 6.40 (s, 1H), 5.64 (d, J=10 Hz, 1H), 5.47 (d, J=9.5 Hz, 1H),5.15 (m, 1H), 5.03 (d, J=6 Hz, 1H), 2.68 (t, J=9.5 Hz, 2H), 2.41 (t, J=7Hz, 2H); LC/MS 913 (M−H).

Diacetylmorphine (1, 242 mg/0.66 mmol) was added to an oven dried flaskequipped with a magnetic stir bar followed by the addition of toluene (6mL), potassium carbonate (182 mg/1.84 mmol) and trichloroethylchloroformate (0.36 mL/2.64 mmol) at room temperature. The resultingsuspension was then refluxed at 120° C. for 20 hours. After this timeperiod, LC/MS indicated starting material remained. The solution wasthen cooled to room temp, additional trichloroethyl chloroformate wasadded (0.20 mL/1.47 mmol), and the solution was refluxed at 120° C. for8 hours. After this time period the reaction was complete, as monitoredby LC/MS. Potassium carbonate was then removed by filtration and toluenewas removed under reduced pressure. To the resulting residue was addedTHF (0.20 mL) and 90% acetic acid (0.105 mL), and the suspension wasstirred at room temperature for 6 hours, after which time the reactionwas complete as monitored by LC/MS. The solution was separated from themajority of the solid Zn by pipette, and filtered through coarse filterpaper into a separatory funnel. The filter paper was washed withisopropanol (5 mL), CHCl₃ (40 mL) and H₂O (20 mL). The aqueous layer wassaturated with NaCl, followed by the addition of 50% aqueous NH₂OHdropwise until the pH of the solution reached approximately 7 (pHpaper). During this time the funnel was shaken periodically to promoteextraction of 18 into the organic phase. CHCl₃ was then separated, and anew portion of CHCl₃ (20 mL) was added before the pH of the solution wasadjusted to approximately 9 using 50% aqueous NH₂OH. The separatoryfunnel was shaken during the pH adjustment periodically. The organiclayer was separated, and the resulting aqueous solution was extractedwith CHCl₃ (3×15 mL). The combined organic layers were dried with MgSO₄,and the solvents removed under reduced pressure to yield the crudeproduct as a yellow oil (233 mg/100% crude yield); LC/MS 356 (M+H⁺). Thecrude product was used in the next step without further purification.

Nordiacetylmorphine (18) (65 mg/0.18 mmol) was dissolved in anhydrousCH₂Cl₂ (3 mL), and the solution was cooled to 0° C., followed by theaddition of DIPEA (0.063 mL/0.36 mmol) and 3-chloropropylsulfonylchloride (0.024 mL/0.20 mmol) at 0° C. The reaction was stirred at 0° C.for one hour, then allowed to warm to room temperature with stirringovernight. H₂O (4 mL) and EtOAc (10 mL) were then added. The layers wereseparated, and the aqueous layer was extracted with EtOAc (2×5 mL). Thecombined organic layers were washed with H₂O (1×5 mL) and brine (1×5mL), dried over MgSO₄ and the solvent removed under reduced pressure.The crude product was purified by preparative TLC (1:2 Hexanes:EtOAc) togive 19 as an amorphous solid (40 mg/44% yield). ¹H NMR (500 MHz, CDCl₃)δ 6.82 (d, J=8.2 Hz, 1H), 6.62 (d, J=8.2 Hz, 1H), 5.71 (d, J=11.4 Hz,1H), 5.42 (d, J=10.3 Hz, 1H), 3.71 (t, J=5.9 Hz, 2H), 2.28 (s, 3H), 2.14(s, 3H); LC/MS 495 (M−H).

Chloride 19 (128 mg/0.26 mmol) was dissolved in anhydrous DMF (4 mL)followed by the addition of potassium thioacetate (148 mg/1.30 mmol),and the solution was heated to 90° C. for 5 hours. The reaction wascooled to room temperature, EtOAc (15 mL) was added, and the solutionwas transferred to a separatory funnel. The organic phase was washedwith H₂O (1×5 mL) and brine (2×5 mL), dried with MgSO₄ and the solventsremoved under reduced pressure to yield the crude product which waspurified by ISCO (20:1 Hexanes:EtOAc to 5:1 Hexanes:EtOAc) to givethioacetate 20 as an amorphous solid (85 mg/62% yield). ¹H NMR (500 MHz,CDCl₃) δ 6.81 (d, J=9.6 Hz, 1H), 6.61 (d, J=8.3 Hz, 1H), 5.71 (d, J=10.0Hz, 1H), 5.42 (d, J=10.1 Hz, 1H), 3.06 (t, J=7.1 Hz, 2H), 2.36 (s, 3H),2.28 (s, 3H); LC/MS 534 (M−H).

Thioacetate 20 (29 mg/0.054 mmol) was dissolved in CH₃OH/THF (2 mL:0.6mL), followed by the addition of 100 mM sodium phosphate buffer (pH7.0). 15 mg of hydroxylamine hydrochloride was then added, and theresulting solution was stirred overnight at room temperature. Thecontents of the vial were transferred to a separatory funnel, EtOAc (10mL) and water (5 mL) were added, and the funnel was shaken. The layerswere separated, and the aqueous layer was extracted with EtOAc (1×5 mL).The combined organic layers were washed with brine (1×5 mL), dried withMgSO₄ and the solvent removed under reduced pressure. The crude productwas purified by preparative HPLC to give the pure product as thedisulfide derivative of 21 (LC/MS) (2.6 mg/10% yield). Free thiol 21 wasobtained by dissolving the disulfide in DMSO:sodium phosphate buffer (pH7.5) (400 uL DMSO:440 uL 50 mM NaPi), followed by the addition of TCEP(2.2 mg/0.008 mmol) in one portion. The resulting solution was stirredat room temperature for 30 minutes to yield 21. LC/MS 452 (M−H).

Diacetylmorphine 1 (10 mg, 0.03 mmol) was dissolved in acetonitrile (1mL). To the mixture was added 2-bromo-N-acetyl-HCTL (16 mg, 0.067 mmol).The reaction mixture was then heated at 60° C. for overnight. Afterovernight, the reaction mixture was cooled down to room temperature. Themixture was then purified using silica TLC plate, eluted with 10%MeOH/DCM to afford 3.6 mg (22%) of 22 as a white powder. ¹H NMR(DMSO-d6) δ 9.14 (1H, d), 6.88 (1H, d), 6.71 (1H, d), 5.65 (1H, d), 5.53(1H, d); LC/MS 528 (M+).

Example 2. Conjugates

A 6-AM derivative 23 having a linking group containing a sulfhydrylgroup reactive with maleimide or haloacetyl or haloacetamido moiety:

was used to prepare conjugates to a latex solid phase and to KLH asdescribed below.

Bovine serum albumin (“BSA”) and polystyrene latex particles(Interfacial Dynamics) were incubated at 25° C. for 30 minutes at 1-10mg BSA per mL of latex slurry at 1-10% solids in 25 mM citrate buffer,pH approximately 4. The solution was then brought to approximatelyneutral pH with 150 mM potassium phosphate/30 mM potassium borate, andincubated for an additional 2 hours at 25° C. The suspension was washedthree times by resuspension in 50 mM potassium phosphate/10 mM potassiumborate/150 mM sodium chloride at approximately neutral pH followed bycentrifugation.

An N-hydroxysuccinimide/maleimide bifunctional poly(ethylene glycol)crosslinker as described in U.S. Pat. No. 6,887,952 was added at 5-500mg/mL in deionized water to the BSA-latex particles at 1-10% solids. Thecrosslinker was incubated with the BSA-latex particles at roomtemperature for 2 hours. Excess crosslinker was removed bycentrifugation and resuspension in PBS of the nowmaleimide-functionalized BSA-latex particles.

The derivative (4-8 mg) was dissolved in 0.8 mL DMF-water solution(70:30 v/v) and 200 μL of 1 M KOH, and was incubated for 10 minutes atroom temperature. Then the excess of the base was neutralized with aphosphate/hydrochloric acid buffer to pH 7. Maleimide-functionalizedBSA-latex particles were added to the solution containing the 6-AMderivative in the presence of 0.1 mM EDTA, and the mixture was incubatedat room temperature overnight. KOH was added to maintain the pH at about7.0. The reaction was stopped in two steps. First by addition of 0.2 mMβ-mercaptoethanol and incubation for 30 at room temperature and then byaddition of 6 mM N-(hydroxyethyl)maleimide and additional incubation for30 minutes at room temperature. The 6-AM derivative-conjugated latexparticles were purified by centrifugation and resuspension in PBS.

Keyhole Limpet Hemocyanin (KLH, Calbiochem #374817, 50 mg/mL inglycerol) was passed through a 40 mL GH25 column equilibrated in 0.1Mpotassium phosphate, 0.1M borate, 0.15M sodium chloride buffer, pH 7.5to remove glycerol. A 1.5-fold molar excess of N-ethylmaleimide wasadded, and the mixture incubated 30 minutes at room temperature. A200-fold molar excess of sulfo-SMCC (Pierce #22322) from a 50 mM stockin distilled water was added while vortexing. Vortexing was continuedfor another 30 seconds, followed by incubation for 10 minutes at roomtemperature. A 100-fold molar excess of SMCC (Pierce #22360) from an 80mM stock in acetonitrile was added while vortexing. 1M KOH was added tomaintain a pH of between 7.2 and 7.4. The mixture was stirred at roomtemperature for 90 minutes. After 90 minutes incubation, KLH-SMCC waspurified by gel filtration using a GH25 column equilibrated in 0.1Mpotassium phosphate, 0.02M borate, 0.15M sodium chloride buffer, pH 7.0.

The 6-AM derivative (4-8 mg) was dissolved in 0.8 mL DMF-water solution(70:30 v/v) and 200 μL of 1 M KOH, and was incubated for 10 minutes atroom temperature. The excess of the base was neutralized with aphosphate/hydrochloric acid buffer and pH brought to 7. Then, a 2-foldmolar excess of derivative (based on the concentration of SMCC in aparticular batch of KLH-SMCC) was added to KLH-SMCC, and the mixturestirred for 90 minutes at room temperature. Conjugates were purified byexhaustive dialysis in PBS.

Example 3. Antibodies

Following immunization with the KLH-conjugated derivative, phage displayantibody libraries were constructed and enriched using biotin-conjugated24 and magnetic streptavidin latex as generally described in U.S. Pat.No. 6,057,098. The antibody phage library was selected with 24,transferred into a plasmid expression vector and electro-porated intobacterial cells. Simultaneous negative selection was performed with 25,26, and 27 to select against antibodies binding to undesired epitopes.

The bacterial cells from each antibody library were streaked on agar togenerate colonies. The colonies, coding for monoclonal antibodies, wereused to inoculate culture medium in individual wells in 96-well plates.The liquid cultures were grown overnight and used to generate frozencell stocks. The frozen cell stocks were used to generate duplicate96-well plate cultures, followed by expression and purification of themonoclonal antibodies in soluble form in microgram quantities. Acompetitive assay for 6-AM developed with a selected antibody exhibitedno crossreactivity with morphine, morphine-3-glucuronide, ormorphine-6-glucuronide at clinically relevant concentrations.

Example 4. Cross Reactivity

Cross reactivity to heroin metabolites and other common structurallyrelated opiates were evaluated using the antibodies as described withreference to Example 3. Immunoassays were constructed using theantibodies of Example 3, configured to operate in a competitive modeimmunoassay format, in which the analogue compounds of the inventionwere compared with other related compounds for cross reaction against6-acetylmorphine. Labeled 6-acetylmorphine conjugates were prepared foruse as the detectable species. Aliquots of labeled 6-acetylmorphine at10 ng/mL were incubated in the presence of competing compound with theantibody of the invention, and the level of interaction of thecompetitor compound determined as a reduction in measured signalcompared with the situation where only 6-acetylmorphine was present. Theresults are provided in the Table 1. The data demonstrate the highspecificity of the antibody for 6-acetylmorphine. With many of thecompeting species being applied at an excess of 100,000 to 1 over6-acetylmorphine; there was no detectable cross reaction; and with6-acetylcodeine at a 30:1 excess over 6-acetylmorphone there was only a3% cross reaction. The data clearly indicate the specificity of theantibodies of the invention for 6-acetylmorphine.

The data shown in FIG. 7 indicate the behaviors of several6-actylmorphone conjugates when incubated with the antibody of theinvention in the presence of increasing 6-acetylmorphine concentrations,indicating the reduction in signal as the 6-acetylmorphine conjugate isdisplaced from the antibody by native 6-acetylmorphine.

TABLE 1 Cross- Analyte Conc. 6-AM Conc. Reactivity Compound (ng/mL)(ng/mL) (%) 6-Acetylmorphine 10 10 100 6-Acetylcodeine 300 10 3 Codeine1,000,000 10 Not Detectable Heroin 750 10 1.3 Hydrocodone 1,000,000 10Not Detectable Hydromorphone 300,000 10 Not Detectable Morphine1,000,000 10 Not Detectable Morphine 1,000,000 10 Not 3-D-glucuronideDetectable Morphine 1,000,000 10 Not 6-D-glucuronide DetectableOxycodone 1,000,000 10 Not Detectable Oxymorphone 350,000 10 NotDetectable

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.Modifications therein and other uses will occur to those skilled in theart. These modifications are encompassed within the spirit of theinvention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

We claim:
 1. A compound of formula:

or a salt thereof.
 2. A conjugate comprising the compound of claim 1covalently bound to a protein, polypeptide, detectable label, nucleicacid, or solid phase, wherein the sulfhydryl moiety of the compound isconjugated to a corresponding coupling site on the protein, polypeptide,detectable label, nucleic acid, or solid phase.
 3. The conjugate ofclaim 2, wherein the compound is covalently bound to the detectablelabel, and the detectable label is selected from the group consisting ofan enzyme, a fluorophore, biotin, avidin, streptavidin, digoxigenin,maltose, oligohistidine, 2,4-dinitrobenzene, phenylarsenate, and afluorescent latex particle.
 4. The conjugate of claim 2, wherein thecompound is covalently bound to the protein, and the protein is keyholelimpet hem ocyanin or bovine serum albumin.
 5. The conjugate of claim 2,wherein the compound is covalently bound to the solid phase, and thesolid phase is selected from the group consisting of a membrane, acellulose-based paper, a polymeric particle, a latex particle, aparamagnetic particle, a glass substrate, a silicon substrate, a plasticsubstrate, and a multiple-well plate.
 6. The conjugate of claim 2,wherein the conjugate further comprises a cross-linker.
 7. The conjugateof claim 6, wherein the cross-linker comprises a polyethylene glycolpolymer.
 8. A method of preparing a conjugate, comprising: contactingthe compound of claim 1 with a protein, polypeptide, detectable label,nucleic acid, or solid phase under conditions selected to providecovalent coupling of said compound to said protein, polypeptide,detectable label, nucleic acid, or solid phase through the sulfhydrylreactive moiety of the compound.
 9. The method of claim 8, furthercomprising introducing said one or more coupling sites corresponding tothe sulfhydryl reactive moiety into said protein, polypeptide,detectable label, nucleic acid, or solid phase prior to said contactingstep.
 10. The method of claim 9, wherein the introducing step comprisescoupling of the protein, polypeptide, detectable label, nucleic acid, orsolid phase to one or more bivalent crosslinkers.
 11. A method ofstimulating an immune response to 6-acetylmorphine (6-AM), comprising:immunizing an animal with a conjugate of claim
 4. 12. The method ofclaim 11, further comprising isolating one or more antibodies thatspecifically bind 6-AM.
 13. The method of claim 12, wherein the one ormore antibodies have a binding affinity for 6-AM of at least a factor of30 greater than the affinity of the antibody for 6-acetylcodeine andheroin, at least a factor of 30,000 greater than the affinity of theantibody for hydromorphone and oxymorphone, and at least a factor of100,000 greater than the affinity of the antibody for codeine,hydrocodone, morphine, morphine 3-D-glucuronide, morphine6-D-glucuronide, and oxycodone.
 14. The method of claim 12, wherein saidone or more antibodies are isolated directly from said animal.
 15. Amethod of determining a 6-acetylmorphine (6-AM) concentration in asample, comprising: performing a competitive binding assay using aconjugate according to claim 3 which competes with 6-AM in said samplefor binding to an antibody, wherein the conjugate comprises a detectablelabel covalently bound thereto, wherein a signal obtained fromdetermining an amount of detectable label bound to the antibody in saidassay is indicative of the concentration of 6-AM in said sample; anddetermining the concentration of 6-AM in said sample from the assaysignal.